Flow through electroporation instrumentation

ABSTRACT

The present disclosure provides a flow-through electroporation device configured for use as a stand-alone module or as one module in an automated multi-module processing system.

RELATED APPLICATIONS

This application claims priority to U.S. Patent Application Ser. No.62/566,374, entitled “Electroporation Device,” filed Sep. 30, 2017; U.S.Patent Application Ser. No. 62/566,375, entitled “ElectroporationDevice,” filed Sep. 30, 2017; U.S. Patent Application Ser. No.62/566,688, entitled “Introduction of Exogenous Materials into Cells,”filed Oct. 2, 2017; U.S. Patent Application Ser. No. 62/567,697,entitled “Automated Nucleic Acid Assembly and Introduction of NucleicAcids into Cells,” filed Oct. 3, 2017; U.S. Patent Application Ser. No.62/620,370, entitled “Automated Filtration and Manipulation of ViableCells,” filed Jan. 22, 2018; U.S. Patent Application Ser. No.62/649,731, entitled “Automated Control of Cell Growth Rates forInduction and Transformation,” filed Mar. 29, 2018; U.S. PatentApplication Ser. No. 62/671,385, entitled “Automated Control of CellGrowth Rates for Induction and Transformation,” filed May 14, 2018; U.S.Patent Application Ser. No. 62/648,130, entitled “Genomic Editing inAutomated Systems,” filed Mar. 26, 2018; U.S. Patent Application Ser.No. 62/657,651, entitled “Combination Reagent Cartridge andElectroporation Device,” filed Apr. 13, 2018; U.S. Patent ApplicationSer. No. 62/657,654, entitled “Automated Cell Processing SystemsComprising Cartridges,” filed Apr. 13, 2018; and U.S. Patent ApplicationSer. No. 62/689,068, entitled “Nucleic Acid Purification Protocol forUse in Automated Cell Processing Systems,” filed Jun. 23, 2018. Allabove identified applications are hereby incorporated by reference intheir entireties for all purposes.

BACKGROUND OF THE INVENTION

In the following discussion certain articles and methods will bedescribed for background and introductory purposes. Nothing containedherein is to be construed as an “admission” of prior art. Applicantexpressly reserves the right to demonstrate, where appropriate, that thearticles and methods referenced herein do not constitute prior art underthe applicable statutory provisions.

The cell membrane constitutes the primary barrier for the transport ofmolecules and ions between the interior and the exterior of a cell.Electroporation, also known as electropermeabilization, substantiallyincreases cell membrane permeability in the presence of a pulsedelectric field. Traditional electroporation systems have been widelyused; however, traditional systems require high voltage input and sufferfrom adverse environmental conditions such as electric field distortion,local pH variation, metal ion dissolution and excess heat generation,all of which may contribute to low electroporation efficiency and/orcell viability. Further, traditional electroporation systems are noteasily automated or incorporated into automated cell processing systemswhere electroporation is but one process performed. There is thus a needfor automated multi-module cell processing systems and componentsthereof capable of transforming multiple cells in an efficient andautomated fashion. The present invention addresses this need.

SUMMARY OF THE INVENTION

This Summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This Summary is not intended to identify key or essentialfeatures of the claimed subject matter, nor is it intended to be used tolimit the scope of the claimed subject matter. Other features, details,utilities, and advantages of the claimed subject matter will be apparentfrom the following written Detailed Description including those aspectsillustrated in the accompanying drawings and defined in the appendedclaims.

The present disclosure provides an electroporation device configuredboth for use as a stand-alone electroporation device and for use in anautomated multi-module cell processing environment. The device comprisesa flow-through electroporation (FTEP) device for introducing exogenousmaterials into cells in a liquid medium, where the device comprises aninlet and an inlet channel for introducing cells and exogenous materialsinto the FTEP device, an outlet channel and an outlet for removingtransformed cells from the FTEP device, a flow channel positionedbetween the inlet and outlet channels where the flow channel optionallydecreases in width between the point where the inlet channel enters theflow channel and the outlet channel exits the flow channel, and twoelectrodes. In some embodiments, the two electrodes form a portion ofthe wall of the flow channel where the flow channel decreases in width.In other embodiments, the electrodes may be positioned such that a firstelectrode channel fluidically connects the first electrode to the flowchannel between the inlet channel and the narrow portion of the flowchannel, and a second electrode channel fluidically connects the secondelectrode to the flow channel between the narrow portion of the flowchannel and the outlet channel.

Thus, in certain embodiments a flow-through electroporation (FTEP)device for introducing an exogenous material into cells in a fluid isprovided, where the FTEP device comprises at least a first inlet and atleast a first inlet channel for introducing a fluid comprising cells andexogenous material into the FTEP device; an outlet and an outlet channelfor removing a fluid comprising transformed cells and exogenous materialfrom the FTEP device; a flow channel intersecting and positioned betweenthe first inlet channel and the outlet channel, wherein the flow channeldecreases in width between the first inlet channel and the center of theflow channel and the outlet channel and the center of the flow channel;and two electrodes positioned in electrode channels between theintersection of the flow channel with the first inlet channel and theintersection of the flow channel with the outlet channel and on eitherend of where the flow channel decreases in width; wherein the electrodesare in fluid communication with fluid in the flow channel; and whereinthe electrodes apply one or more electric pulses to the cells in thefluid as they pass through the flow channel, thereby introducing theexogenous material into the cells in the fluid.

In some aspects of this embodiment, the FTEP device further comprises areservoir connected to the at least first inlet for introducing thecells in fluid into the FTEP device and a reservoir connected to theoutlet for removing transformed cells from the FTEP device. In someaspects of this embodiment, the reservoirs coupled to the inlet(s) andoutlet range in volume from 100 μL, to 10 ml, or from 0.5 ml to 7 ml, orfrom 1 ml to 5 ml.

In some aspects of this embodiment, the FTEP device comprises two inletsand two inlet channels and further comprises a reservoir connected tothe second inlet for introducing the exogenous material into the FTEPdevice. In some configurations of this aspect, the second inlet andsecond inlet channel are located between the first inlet and first inletchannel and the electrodes; and in some configurations, the second inletand second inlet channel are located between the electrodes and theoutlet channel and outlet.

In some aspects of this embodiment, the two electrodes in the FTEPdevice are located from 0.5 mm to 10 mm apart, or from 1 mm to 8 mmapart, or from 3 mm and 7 mm apart, or from 4 mm to 6 mm apart. In someaspects of this embodiment, the FTEP device is between 3 cm to 15 cm inlength, or between 4 cm to 12 cm in length, or from 4.5 cm to 10 cm inlength, or from 5 cm to 8 cm in length. In some aspects of thisembodiment, this embodiment of the FTEP device is between 0.5 cm to 5 cmin width, or from 0.75 cm to 3 cm in width, or from 1 cm to 2.5 cm inwidth, or from 1 cm to 1.5 cm in width. In some aspects of thisembodiment, the narrowest part of the channel width in the FTEP deviceis from 10 μM to 5 mm.

In some aspects of this embodiment, the flow rate in the FTEP rangesfrom 0.1 ml to 5 ml per minute, or from 0.5 ml to 3 ml per minute, orfrom 1 ml to 2.5 ml per minute.

In some aspects of this embodiment the electrodes are configured todeliver 1-25 Kv/cm, or 10-20 Kv/cm.

In some aspects of this embodiment, the FTEP device further comprisesone or more filters between the one or more inlet channels and theoutlet channel. In some aspects, there are two filters, one between theinlet channel and the narrowed portion of the flow channel, and onebetween the narrowed portion of the flow channel and the outlet channel.In some aspects of this embodiment, the filters are graduated in poresize with the larger pores proximal to the inlet chamber or outletchamber, and the small pores proximal to the narrowed portion of theflow channel. In some aspects, the small pores are the same size orlarger than the size of the narrowed portion of the flow channel. Insome aspects of this embodiment, the filter is formed separately fromthe body of the FTEP device and placed into the FTEP device as it isbeing assembled. Alternatively, in some aspects of this embodiment, thefilter may be formed as part of and integral to the body of the FTEPdevice.

In some aspects of this embodiment, the FTEP devices can provide a celltransformation rate of 10³ to 10¹² cells per minute, or 10⁴ to 10¹⁰ perminute, or 10⁵ to 10⁹ per minute, or 10⁶ to 10⁸ per minute. Typically,10⁸ yeast cells may be transformed per minute, and 10¹⁰-10¹¹ bacterialcells may be transformed per minute.

In some aspects of this embodiment, the transformation of cells resultsin at least 90% viable cells, or 95% viable cells, and up to 99% viablecells.

In some aspects of this embodiment, the FTEP device is manufactured byinjection molding from crystal styrene, cyclo-olefin polymer, orcyclo-olefin co-polymer, and in some aspects of this embodiment theelectrodes are fabricated from stainless steel.

Yet another embodiment provides a flow-through electroporation (FTEP)device for introducing an exogenous material into cells in a fluid, thedevice comprising: at least one inlet and at least one inlet channel forintroducing a fluid comprising cells and exogenous material to the FTEPdevice; an outlet and an outlet channel for removing transformed cellsand exogenous material from the FTEP device; a flow channel positionedbetween a first inlet channel and the outlet channel where the flowchannel intersects with the first inlet channel and the outlet channeland where a portion of the flow channel narrows between the inletchannel intersection with the flow channel and the outlet channelintersection with the flow channel; and an electrode positioned oneither side of the flow channel and in direct contact with the fluid inthe flow channel, and where the electrodes define the narrowed portionof the flow channel; and wherein the electrodes apply one or moreelectric pulses to the cells in the fluid as they pass through the flowchannel, thereby introducing the exogenous material into the cells inthe fluid.

In some aspects, the FTEP device further comprises a reservoir connectedto the inlet for introducing the cells in fluid into the FTEP device anda reservoir connected to the outlet for removing transformed cells fromthe FTEP device, and in some aspects, the FTEP device comprises twoinlets and two inlet channels and further comprises a reservoirconnected to a second inlet for introducing the exogenous material intothe FTEP device. In some aspects of this embodiment, the reservoirscoupled to the inlet(s) and outlet range in volume from 100 μL, to 10ml, or from 0.5 ml to 7 ml, or from 1 ml to 5 ml.

In some aspects of this embodiment, the second inlet and second inletchannel are located between the first inlet and first inlet channel andthe electrodes, and in some aspects the second inlet and second inletchannel are located between the electrodes and the outlet channel andoutlet.

In some aspects of this embodiment, the two electrodes in the FTEPdevice are located from 10 μm to 1 mm apart, or from 25 μm to 3 mmapart, or from 50 μm and 2 mm apart, or from 75 μm to 1 mm apart. Insome aspects of this embodiment, the FTEP device is between 3 cm to 15cm in length, or between 4 cm to 12 cm in length, or between 4.5 cm to10 cm in length, or between 0.5 cm to 8 cm in length. In some aspects ofthis embodiment, the FTEP device is between 0.5 cm to 5 cm in width, orfrom 0.75 cm to 3 cm in width, or from 1 cm to 2.5 cm in width, or from1 cm to 1.5 cm in width. In some aspects of this embodiment, thenarrowest part of the channel width of the FTEP device is from 10 μM to5 mm, and in some aspects the narrowest part of the channel width isfrom 10 μM to 1 mm.

In some aspects of this embodiment, the flow rate in the FTEP rangesfrom 0.1 ml to 5 ml per minute, or from 0.5 ml to 3 ml per minute, orfrom 1 ml to 2.5 ml per minute.

In some aspects of this embodiment the electrodes are configured todeliver 1-25 Kv/cm, or 10-20 Kv/cm.

In some aspects of this embodiment, the FTEP device further comprisesone or more filters between the one or more inlet channels and theoutlet channel. In some aspects, there are two filters, one between theinlet channel and the narrowed portion of the flow channel, and onebetween the narrowed portion of the flow channel and the outlet channel.In some aspects of this embodiment, the filters are graduated in poresize with the larger pores proximal to the inlet chamber or outletchamber, and the small pores proximal to the narrowed portion of theflow channel. In some aspects, the small pores are the same size orlarger than the size of the narrowed portion of the flow channel. Insome aspects of this embodiment, the filter is formed separately fromthe body of the FTEP device and placed into the FTEP device as it isbeing assembled. Alternatively, in some aspects of this embodiment, thefilter may be formed as part of and integral to the body of the FTEPdevice.

In some aspects of this embodiment, the FTEP devices can provide a celltransformation rate of 10³ to 10¹² cells per minute, or 10⁴ to 10¹⁰ perminute, or 10⁵ to 10⁹ per minute, or 10⁶ to 10⁸ per minute. Typically,10⁸ yeast cells may be transformed per minute, and 10¹⁰-10¹¹ bacterialcells may be transformed per minute.

In some aspects of this embodiment, the transformation of cells resultsin at least 90% viable cells, or 95% viable cells, and up to 99% viablecells.

In some aspects of this embodiment, the FTEP device is manufactured byinjection molding from crystal styrene, cyclo-olefin polymer, orcyclo-olefin co-polymer, and in some aspects the electrodes arefabricated from stainless steel.

In some aspects of either embodiment, the FTEP devices are fabricated asmultiple FTEP devices in parallel on a single substrate where the FTEPdevices are then separated for use.

In some aspects of either embodiment, the FTEP device is part of areagent cartridge, and in some aspects the reagent cartridge comprises ascript providing operating instructions for the FTEP device.

In some aspects of either embodiment, the FTEP device is comprised in amodule that is part of an automated multi-module cell processing system,wherein the automated multi-module cell processing system comprises oneor more of a receptacle for cells, one or more receptacles for nucleicacids or other material to be electroporated into the cells, a growthmodule, a filtration module, a recovery module, a nucleic acid assemblymodule, a purification module, an editing module, a singulation andgrowth module, a selection module, storage module, and a processor.

In addition, there is presented another embodiment of a flow-throughelectroporation (FTEP) device for introducing an exogenous material intocells in a fluid, the FTEP device comprising: at least a first inlet forreceiving a fluid comprising cells or exogenous materials; a flowchannel having a narrowed region area fluidically coupled to the firstinlet for receiving the fluid, wherein the narrowed region confines aflow the fluid; at least two electrodes positioned in electricalcommunication with the fluid in the narrowed region of the flow channel,the electrodes being configured to apply an electric field to the cellsor exogenous materials as they traverse the narrowed region of the flowchannel, the electric field electroporating the cells thereby formingtransformed cells; and at least one outlet coupled to the narrowedregion of the flow channel and configured to receive the transformedcells. In some aspects, the at least two electrodes are configured to bein direct contact with the fluid. In some aspects of this embodiment theflow channel follows a non-linear path that imparts momentum to thecells such that at least a portion of the cells flow around an objectpositioned in the channel. In some aspects, the FTEP device furthercomprises at least one aperture proximate to the narrowed region of theflow channel for receiving a proximal end of a first electrode, wherebythe first electrode is placed in direct contact with the fluid.Moreover, in some aspects, the aperture comprises a rounded edge. Insome aspects of this embodiment, the narrowed region of the flow channelhas a first cross sectional dimension and the at least two electrodesextend only partially across the narrowed region of the flow channelalong that dimension.

As with the other embodiments, the FTEP device further comprises areservoir connected to the inlet for introducing the cells in fluid intothe FTEP device and a reservoir connected to the outlet for removingtransformed cells from the FTEP device.

In some aspects of all embodiments, the FTEP device further comprises aseal to permit pressurization of FTEP device and provision of apressure-driven flow. In some aspects, the FTEP device further comprisesa pump to drive the fluid through the flow channel such that at least aplurality of the cells traverse the narrowed region of the flow channelmore than once.

In some aspects of all embodiments, the FTEP device further comprises avoltage supply to apply time-varying voltage to the at least twoelectrodes.

As with earlier-described embodiments, one aspect of this embodiment ofan FTEP further comprises a first reservoir connected to the first inletfor receiving and retaining the fluid and a second reservoir connectedto the outlet for receiving the transformed cells, and in some aspects,the FTEP device further comprises a second inlet to receive exogenousmaterial to be introduced into cells in the FTEP device, said inletbeing in fluid communication with the narrowed area of the flow channel.Also in some aspects, the FTEP device further comprises a filter elementconfigured to prevent transmission of any object substantially largerthan the cells to the narrowed region of the flow channel, and in someaspects of this embodiment, the filter element is integrally formed withthe structure forming the narrowed region of the flow channel, and insome aspects the filter element has progressively smaller apertures atlocations closer to the narrowed region of the flow channel.

In other embodiments, a flow-through electroporation (FTEP) device forintroducing an exogenous material into cells in a fluid is provided, thedevice comprising at least one inlet and at least one inlet channel forintroducing a fluid comprising cells and exogenous material to the FTEPdevice; an outlet and an outlet channel for removing transformed cellsand exogenous material from the FTEP device; a flow channel positionedbetween a first inlet channel and the outlet channel, wherein the flowchannel intersects with the first inlet channel and the outlet channeland wherein a portion of the flow channel narrows between the inletchannel intersection and the outlet channel intersection; and anelectrode positioned on either side of the narrowed portion of the flowchannel and in direct contact with the fluid in the flow channel, theelectrodes defining the narrowed portion of the flow channel; andwherein the electrodes apply one or more electric pulses to the cells inthe fluid as they pass through the flow channel, thereby introducing theexogenous material into the cells in the fluid.

Yet other embodiments provide a flow-through electroporation (FTEP)device for introducing an exogenous material into cells in a fluid, theFTEP device comprising at least a first inlet and at least a first inletchannel for receiving a fluid comprising cells and/or exogenous materialinto the FTEP device; an outlet and an outlet channel for removing afluid comprising transformed cells and exogenous material from the FTEPdevice; a flow channel intersecting and positioned between the at leastfirst inlet channel and the outlet channel; two electrodes positioned inelectrode channels between the intersection of the flow channel with thefirst inlet channel and the intersection of the flow channel with theoutlet channel; wherein the electrodes are in fluid and electricalcommunication with fluid in the flow channel; and wherein the electrodesapply one or more electric pulses to the cells in the fluid as they passthrough the flow channel, thereby introducing exogenous material intothe cells in the fluid; and a filter positioned between the at leastfirst inlet channel and the electrode channels.

In some aspects, the filter is integrally-formed as part of the FTEPdevice, and in some aspects, the filter and FTEP device are formed byinjection-molding. In some aspects, the filter is a gradient filter, andin some aspects, the gradient comprises large pores proximal to the atleast first inlet channel, and small pores proximal to the electrodechannels. In some aspects of FTEP devices with filters, there is asecond filter positioned between the electrodes and the outlet channel,and in some aspects, both filters are integrally formed as part of theFTEP device. In some aspects of this embodiment, both filters aregradient filters, and in some aspects, one gradient filter compriseslarge pores proximal to the at least first inlet channel and small poresproximal to the electrode channels, and wherein the other gradientfilter comprises large pores proximal to the outlet channel and smallpores proximal to the electrode channels.

In some aspects of this embodiment, the flow channel decreases in widthbetween the first inlet channel and the center of the flow channel andthe outlet channel and the center of the flow channel, and in someaspects of this embodiment, the two electrodes are positioned inelectrode channels between the intersection of the flow channel with thefirst inlet channel and the intersection of the flow channel with theoutlet channel and on either end of where the flow channel decreases inwidth.

In aspects of the embodiments of the FTEP device that comprises afilter, the filter elements include “pegs” or “protrusions”, and the“pegs” or “protrusions” may be round, oval, elliptical, or polygonal inshape.

Yet another embodiment provides a method of electroporating cellscomprising: providing a flow-through electroporation (FTEP) device,wherein the flow-through electroporation device comprises an inlet andat least an inlet channel for receiving a fluid comprisingelectrocompetent cells and exogenous material into the FTEP device; anoutlet and an outlet channel for removing a fluid comprising transformedcells and exogenous material from the FTEP device; a flow channelintersecting and positioned between the inlet channel and the outletchannel; and two electrodes positioned between the intersection of theflow channel with the inlet channel and the intersection of the flowchannel with the outlet channel; wherein the electrodes are in fluid andelectrical communication with fluid in the flow channel; flowing thecells comprising the electrocompetent cells and exogenous material intothe inlet and the inlet channel; flowing the cells through the flowchannel and past the two electrodes; providing electrical pulses to thecells in the fluid as the cells flow through the flow channel past theelectrodes producing electroporated cells; and removing theelectroporated cells from the outlet channel and outlet.

In some aspects the flow channel in the FTEP device decreases in widthbetween the inlet channel and a mid-region of the flow channel and theoutlet channel and the mid-region of the flow channel, and in someaspects of this embodiment, the flow channel decreases in width tobetween 10 μm and 5 mm, or to between 50 μm and 2 mm, or to a dimensionno smaller than at least 2× diameters of the cells being electroporated.

In some aspect, the electrodes are configured to deliver a voltage of1-25 Kv/cm, or a voltage of 5-20 Kv/cm, or a voltage of 10-20 Kv/cm. Insome aspects the flow rate of the FTEP device is between 0.1 mL to 5 mLper minute, or between 0.5 mL to 3 mL per minute.

In some aspects of this embodiment, the two electrodes are each disposedwithin an electrode channel, and one electrode is located between theinlet channel and the mid-region of the flow channel and one electrodeis located between the outlet channel and the mid-region of the flowchannel. In some configurations of this aspect, in the electrodes arebetween 0.5 mm to 10 mm apart, or between 3 mm to 7 mm apart.

In some aspects of this embodiment, the electrodes are positioned oneither side of the flow channel, are in direct contact with the fluid inthe flow channel and define the decrease in width of the flow channel.In some configurations of this aspect, the electrodes are between 10 μmto 5 mm apart, or between 25 μm to 2 mm apart.

In some aspects of this embodiment, the FTEP device further comprises atleast one filter disposed within the flow channel, and in someconfigurations of this aspect, the filter is integrally-formed as partof the FTEP device. In some configurations, the filter is a gradientfilter, and in some configurations, the gradient comprises large poresproximal to the inlet channel, and small pores proximal to theelectrodes. Further, in some configurations the FTEP device furthercomprises a second filter positioned between the electrodes and theoutlet channel, and in some configurations both filters are integrallyformed as part of the FTEP device. Also, in some configurations wherethere are two filters, one gradient filter comprises large poresproximal to the inlet channel and small pores proximal to theelectrodes, and wherein the second gradient filter comprises large poresproximal to the outlet channel and small pores proximal to theelectrodes.

Additionally provided is a method of electroporating cells comprising:providing a flow-through electroporation (FTEP) device, wherein theflow-through electroporation device comprises an inlet and an inletchannel for receiving a fluid comprising electrocompetent cells andexogenous material into the FTEP device; an outlet and an outlet channelfor removing a fluid comprising transformed cells and exogenous materialfrom the FTEP device; a flow channel intersecting and positioned betweenthe inlet channel and the outlet channel; and two electrodes positionedbetween the intersection of the flow channel with the channel and theintersection of the flow channel with the outlet channel; wherein theelectrodes are in fluid and electrical communication with fluid in theflow channel; flowing the cells comprising the electrocompetent cellsand exogenous material into the inlet and the inlet channel; flowing thecells through the flow channel and past the two electrodes; providingelectrical pulses to the cells in the fluid as the cells flow throughthe flow channel past the electrodes producing electroporated cells;flowing the electroporated cells to the outlet channel and outlet;reversing the flow of the cells to flow the cells from the outlet,through the outlet channel, through the flow channel and past the twoelectrodes; providing electrical pulses to the cells in the fluid as thecells flow through the flow channel; and flowing the electroporatedcells into the inlet channel and inlet.

Some aspects of this embodiment further comprise, after the flowing theelectroporated cells into the inlet step, again reversing the flow ofthe cells to flow the cells from the inlet, through the inlet channel,through the flow channel and past the two electrodes, providingelectrical pulses to the cells in the fluid as the cells flow throughthe flow channel past the two electrodes; and removing theelectroporated cells from the outlet channel and outlet.

In some aspects of this embodiment, the flow channel in the FTEP devicedecreases in width between the inlet channel and a mid-region of theflow channel and the outlet channel and the mid-region of the flowchannel, and in some aspects the flow channel decreases in width to adimension no smaller than at least 2× diameters of the cells beingelectroporated.

These aspects and other features and advantages of the invention aredescribed below in more detail.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the present inventionwill be more fully understood from the following detailed description ofillustrative embodiments taken in conjunction with the accompanyingdrawings in which:

FIG. 1A is an illustration of a top view of one aspect of the FTEPdevices of the disclosure. FIG. 1B is an illustration of the top view ofa cross section of the aspect of the device shown in FIG. 1A. FIG. 1C isan illustration of a side view of a cross section of the aspect of thedevice shown in FIGS. 1A and 1B.

FIG. 2A is an illustration of a top view of another aspect of the FTEPdevices of the disclosure. FIG. 2B is an illustration of the top view ofa cross section of the aspect of the device shown in FIG. 2A. FIG. 2C isan illustration of a side view of a cross section of the aspect of thedevice shown in FIGS. 2A and 2B.

FIG. 3A is an illustration of a top view of yet another aspect of theFTEP devices of the disclosure. FIG. 3B is an illustration of the topview of a cross section of the aspect of the device shown in FIG. 3A.FIG. 3C is an illustration of a side view of a cross section of theaspect of the device shown in FIGS. 3A and 3B.

FIG. 4A is an illustration of the top view of a cross section of afurther aspect of the FTEP devices described herein with separate inletsfor the cells and the exogenous materials. FIG. 4B is an illustration ofthe top view of a cross section of the aspect of the device shown inFIG. 4A. FIG. 4C is an illustration of a side view of a cross section ofthe aspect of the device shown in FIG. 4C. FIG. 4D is an illustration ofa side view of a cross section of a variation on the aspect of thedevice shown in FIG. 4D. FIG. 4E is an illustration of a side view of across section of another variation on the aspect of the device shown inFIGS. 4C and 4D.

FIG. 5A is an illustration of the top view of a cross section of yetanother aspect of the FTEP devices of the disclosure where the FTEPcomprises two separate inlets for the cells and the exogenous materials.FIG. 5B is an illustration of a side view of a cross section of theaspect of the device shown in FIG. 5A. FIG. 5C is an illustration of aside view of a cross section of the aspect of the device shown in FIGS.5A and 5B.

FIG. 6 is an illustration of a top view of a cross section of yet anadditional aspect of the FTEP devices of the disclosure, here includingflow focusing of fluid from the input channels.

FIG. 7A is an illustration of a top view of a cross section of a firstmultiplexed aspect of the FTEP devices of the disclosure. FIG. 7B is anillustration of a top view of a cross section of a second multiplexedaspect of the devices of the disclosure. FIG. 7C is an illustration of atop view of a cross section of a third multiplexed aspect of the devicesof the disclosure. FIG. 7D is an illustration of a top view of a crosssection of a fourth multiplexed aspect of the devices of the disclosure.FIG. 7E is an illustration of a top view of a cross section of a fifthmultiplexed aspect of the devices of the disclosure.

FIG. 8A is an illustration of a top view of yet another aspect of theFTEP devices of the disclosure. FIG. 8B is an illustration of the topview of a cross section of the aspect of the device shown in FIG. 8A.FIG. 8C is an illustration of a side view of a cross section of theaspect of the device shown in FIGS. 8A and 8B. FIG. 8D is anillustration of a side view of a cross section of the bottom half of theaspect of the devices shown in FIGS. 8A, 8B and 8C. FIG. 8E is anillustration of a side view of a cross section of a variation of theaspect of the devices shown in FIGS. 8A-8D where here the electrodes arepositioned on the bottom of the FTEP device.

FIG. 9A is an illustration of a top view of yet another aspect of theFTEP devices of the disclosure. FIG. 9B an illustration of the top viewof a cross section of the aspect of the device shown in FIG. 9A. FIG. 9Cis an illustration of a side view of a cross section of the aspect ofthe device shown in FIGS. 9A and 9B.

FIG. 10A is an illustration of a top view of an alternative aspect ofthe FTEP devices of the disclosure. FIG. 10B is an illustration of thetop view of a cross section of the aspect of the device shown in FIG.10A. FIG. 10C is an illustration of the top view of a cross section of avariation of the aspect of the devices shown in FIGS. 10A and 10B.

FIG. 10D is an illustration of a side view of a cross section of theaspects of the devices shown in FIGS. 10A-10C. FIG. 10E is anillustration of a side view of a cross section of the bottom half of theaspects of the devices shown in FIGS. 10A-10D.

FIG. 11A is an illustration of a top view of yet another aspect of theFTEP devices of the disclosure. FIG. 11B is an illustration of the topview of a cross section of the aspect of the device shown in FIG. 11A.FIG. 11C is an illustration of a side view of a cross section of theaspect of the device of the disclosure shown in FIGS. 11A-11B. FIG. 11Dis an illustration of a side view of a cross section of a variation onthe aspect of the device shown in FIGS. 11A-11B. FIG. 11E is anillustration of a side view of a cross section of a variation on theaspect of the device shown in FIGS. 11A-11B.

FIG. 12A is an illustration of the top view of a cross section of yetanother aspect of the FTEP devices of the disclosure. FIG. 12B is anillustration of the top view of a cross section of the aspect of thedevice shown in FIG. 12A. FIG. 12C is an illustration of a side view ofa cross section of the aspect of the device shown in FIGS. 12A and 12B.

FIG. 13A is an illustration of a side view of a cross section of anotheraspect of the FTEP devices of the disclosure. FIG. 13B is anillustration of the top view of a cross section of the aspect of thedevices shown in FIG. 13A.

FIG. 14 is an illustration of a top view of a cross section of an aspectof an FTEP device with a flow focusing feature.

FIGS. 15A through 15C are top perspective, bottom perspective, andbottom views, respectively, of a flow-through electroporation devicethat may be part of a stand-alone FTEP module or as one module in anautomated multi-module cell processing system. FIG. 15D shows scanningelectromicrographs of the FTEP units depicted in FIG. 15C. FIG. 15Eshows scanning electromicrographs of filters 1570 and 1572 depicted asblack bars in FIGS. 15B and 15C. FIG. 15F depicts (i) the electrodesbefore insertion into the FTEP device; (ii) an electrode; and (iii) theelectrode inserted into an electrode channel with the electrode andelectrode channel adjacent to the flow channel. FIG. 15G shows twoscanning electromicrographs of two different configurations of theaperture where the electrode channel meets the flow channel.

FIGS. 16A and 16B depict alternative embodiments of a combinationreagent cartridge and electroporation device.

FIG. 17 depicts an exemplary automated multi-module cell processingsystem comprising an FTEP device and additional optional modules.

FIG. 18 is a block diagram of an embodiment of a method for using anautomated multi-module cell processing system comprising an FTEP in atransformation module.

FIG. 19 is a simplified block diagram of an exemplary automatedmulti-module cell processing system in which one or more of the FTEPdevices described herein may be used.

FIG. 20 is a simplified block diagram of a different embodiment of anexemplary automated multi-module cell processing system in which one ormore of the FTEP devices described herein may be used.

FIG. 21 is a simplified block diagram of yet another embodiment of anexemplary automated multi-module cell processing system in which one ormore of the FTEP devices described herein may be used.

FIG. 22 is a simplified block diagram of an embodiment of an exemplaryautomated multi-module cell processing system in which one or more ofthe FTEP devices described herein may be used.

FIG. 23 is a simplified block diagram of an embodiment of an exemplaryautomated multi-module cell processing system in which one or more ofthe FTEP devices described herein may be used.

FIG. 24 is a photograph demonstrating laminar flow of cells andexogenous material in buffer through a flow channel of an FTEP device ofthe disclosure.

FIG. 25A is a bar graph showing the results of electroporation of E.coli using a device of the disclosure and a comparator electroporationdevice. FIG. 25B is a bar graph showing uptake, cutting, and editingefficiencies of E. coli cells transformed via an FTEP as describedherein benchmarked against a comparator electroporation device.

FIG. 26 is a bar graph showing the results of electroporation of S.cerevisiae using an FTEP device of the disclosure and a comparatorelectroporation method.

FIG. 27 shows a graph of FTEP flow and pressure versus elapsed time(top), as well as a simple depiction of the pressure system and FTEP(bottom).

It should be understood that the drawings are not necessarily to scale,and that like reference numbers refer to like features.

DETAILED DESCRIPTION

All of the functionalities described in connection with one embodimentare intended to be applicable to the additional embodiments describedherein except where expressly stated or where the feature or function isincompatible with the additional embodiments. For example, where a givenfeature or function is expressly described in connection with oneembodiment but not expressly mentioned in connection with an alternativeembodiment, it should be understood that the feature or function may bedeployed, utilized, or implemented in connection with the alternativeembodiment unless the feature or function is incompatible with thealternative embodiment.

The practice of the techniques described herein may employ, unlessotherwise indicated, conventional techniques and descriptions molecularbiology (including recombinant techniques), cell biology, biochemistry,and genetic engineering technology, which are within the skill of thosewho practice in the art. Such conventional techniques and descriptionscan be found in standard laboratory manuals such as Green and Sambrook,Molecular Cloning: A Laboratory Manual. 4th, ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., (2014); Current Protocols inMolecular Biology, Ausubel, et al. eds., (2017); Neumann, et al.,Electroporation and Electrofusion in Cell Biology, Plenum Press, NewYork, 1989; and Chang, et al., Guide to Electroporation andElectrofusion, Academic Press, California (1992), all of which areherein incorporated in their entirety by reference for all purposes.

Note that as used herein and in the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “an oligo” refers toone or more oligos that serve the same function, to “the methods”includes reference to equivalent steps and methods known to thoseskilled in the art, and so forth. That is, unless expressly specifiedotherwise, as used herein the words “a,” “an,” “the” carry the meaningof “one or more.” Additionally, it is to be understood that terms suchas “left,” “right,” “top,” “bottom,” “front,” “rear,” “side,” “height,”“length,” “width,” “upper,” “lower,” “interior,” “exterior,” “inner,”“outer” that may be used herein merely describe points of reference anddo not necessarily limit embodiments of the present disclosure to anyparticular orientation or configuration. Furthermore, terms such as“first,” “second,” “third,” etc., merely identify one of a number ofportions, components, steps, operations, functions, and/or points ofreference as disclosed herein, and likewise do not necessarily limitembodiments of the present disclosure to any particular configuration ororientation.

Additionally, the terms “approximately,” “proximate,” “minor,” andsimilar terms generally refer to ranges that include the identifiedvalue within a margin of 20%, 10% or preferably 5% in certainembodiments, and any values therebetween.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications mentionedherein are incorporated by reference for all purposes, including thepurpose of describing and disclosing devices, formulations andmethodologies that may be used in connection with the presentlydescribed invention.

Where a range of values is provided, it is understood that eachintervening value, between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the invention. The upper and lower limits of thesesmaller ranges may independently be included in smaller ranges, and arealso encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

In the following description, numerous specific details are set forth toprovide a more thorough understanding of the present invention. However,it will be apparent to one of skill in the art that the presentinvention may be practiced without one or more of these specificdetails. In other instances, features and procedures well known to thoseskilled in the art have not been described in order to avoid obscuringthe invention. The terms used herein are intended to have the plain andordinary meaning as understood by those of ordinary skill in the art.

The Invention in General

Electroporation is a widely-used method for permeabilization of cellmembranes that works by temporarily generating pores in the cellmembranes with electrical stimulation. The applications ofelectroporation include the delivery of DNA, RNA, siRNA, peptides,proteins, antibodies, drugs or other substances to a variety of cellssuch as mammalian cells (including human cells), plant cells, archaea,yeasts, other eukaryotic cells, bacteria, and other cell types. Further,mixtures of cell types can also be electroporated in a single run; e.g.,mixtures of E. coli strains, mixtures of bacterial strains, mixtures ofyeast strains, mixtures of mammalian cells. Electrical stimulation mayalso be used for cell fusion in the production of hybridomas or otherfused cells. During a typical electroporation procedure, cells aresuspended in a buffer or medium that is favorable for cell survival. Forbacterial cell electroporation, low conductance mediums, such as water,glycerol solutions and the like, are often used to reduce the heatproduction by transient high current. The cells and material to beelectroporated into the cells (collectively “the cell sample”) is thenplaced in a cuvette embedded with two flat electrodes for an electricaldischarge. For example, Bio-Rad (Hercules, Calif.) makes the GENE PULSERXCELL™ line of products to electroporate cells in cuvettes.Traditionally, electroporation requires high field strength.

Generally speaking, microfluidic electroporation—using cell suspensionvolumes of less than approximately 20 ml and as low as 1 μl—allows moreprecise control over a transfection or transformation process andpermits flexible integration with other cell processing tools comparedto bench-scale electroporation devices. Microfluidic electroporationthus provides unique advantages for, e.g., single cell transformation,processing and analysis; multi-unit FTEP device configurations; andintegrated, automated multi-module cell processing and analysis.

The present disclosure provides electroporation devices, electroporationsystems and methods that achieve high efficiency cell electroporationwith low toxicity where the electroporation devices and systems can beintegrated with other automated cell processing tools. Further, theelectroporation device of the disclosure allows for multiplexing wheretwo to many electroporation units are constructed and used in parallel,which allows for particularly easy integration with robotic liquidhandling instrumentation. Such automated instrumentation includes, butis not limited to, off-the-shelf automated liquid handling systems fromTecan (Mannedorf, Switzerland), Hamilton (Reno, Nev.), Beckman Coulter(Fort Collins, Colo.), etc.

During the electroporation process, it is important to use voltagesufficient for achieving electroporation of material into the cells, butnot too much voltage as too much power will decrease cell viability. Forexample, to electroporate a suspension of a human cell line, 200 voltsis needed for a 0.2 ml sample in a 4 mm-gap cuvette with exponentialdischarge from a capacitor of about 1000 μF. However, if the same 0.2 mlcell suspension is placed in a longer container with 2 cm electrodedistance (5 times of cuvette gap distance), the voltage required wouldbe 1000 volts, but a capacitor of only 40 μF ( 1/25 of 1000 μF) isneeded because the electric energy from a capacitor follows the equationof:

E=0.5 U ² C

where E is electric energy, U is voltage and C is capacitance. Thereforea high voltage pulse generator is easy to manufacture because it needs amuch smaller capacitor to store a similar amount of energy. Similarly,it would not be difficult to generate other wave forms of highervoltages.

The electroporation devices of the disclosure can allow for a high rateof cell transformation in a relatively short amount of time. The rate ofcell transformation is dependent on the cell type and the number ofcells being transformed. For example, for E. coli, the electroporationdevices can provide a cell transformation rate of 10³ to 10¹² cells perminute, 10⁴ to 10¹⁰ per minute, 10⁵ to 10⁹ per minute, or 10⁶ to 10⁸ perminute. Typically, 10⁸ yeast cells may be transformed per minute, and10¹⁰-10¹¹ bacterial cells may be transformed per minute. Theelectroporation devices also allow transformation of batches of cellsranging from 1 cell to 10¹¹ cells in a single transformation procedureusing parallel devices.

Exemplary FTEP Embodiments

A first aspect of the invention described herein is illustrated in FIGS.1A-1C. FIG. 1A shows a planar top view of an FTEP device 100 having aninlet 102 for introducing a fluid containing cells and exogenousmaterial to be delivered to the cells into the FTEP device 100 and anoutlet 104 for removing the transformed cells following electroporation.Oval electrodes 108 are positioned so as to define a center portion ofthe flow channel (not shown) where the channel narrows based on thecurvature of the electrodes. FIG. 1B shows a cutaway view 110 from thetop of the device 100, with the inlet 102, outlet 104, and electrodes108 positioned with respect to a flow channel 106. Note that theelectrodes 108 define a narrowing of flow channel 106. FIG. 1C shows aside cutaway view 120 of the device 100 with the inlet 102 and inletchannel 112, and outlet 104 and outlet channel 114. The electrodes 108are oval in shape and positioned so that they define a narrowed portionof the flow channel 106.

In the FTEP devices of the disclosure, the toxicity level of thetransformation results in greater than 30% viable cells afterelectroporation, preferably greater than 35%, 40%, 45%, 50%, 55%, 60%,70%, 75%, 80%, 85%, 90%, 95%, or even 99% viable cells followingtransformation, depending on the cell type and the nucleic acids beingintroduced into the cells.

The housing of the FTEP device can be made from many materials dependingon whether the FTEP device is to be reused, autoclaved, or isdisposable, including stainless steel, silicon, glass, resin, polyvinylchloride, polyethylene, polyamide, polystyrene, polyethylene,polypropylene, acrylonitrile butadiene, polycarbonate,polyetheretheketone (PEEK), polysulfone and polyurethane, co-polymers ofthese and other polymers. Similarly, the walls of the channels in thedevice can be made of any suitable material including silicone, resin,glass, glass fiber, polyvinyl chloride, polyethylene, polyamide,polyethylene, polypropylene, acrylonitrile butadiene, polycarbonate,polyetheretheketone (PEEK), polysulfone and polyurethane, co-polymers ofthese and other polymers. Preferred materials include crystal styrene,cyclo-olefin polymer (COP) and cyclic olephin co-polymers (COC), whichallow the device to be formed entirely by injection molding in one piecewith the exception of the electrodes and, e.g., a bottom sealing film ifpresent (see, e.g., FIG. 16B(v)).

The FTEP devices described herein (or portions of the FTEP devices) canbe created or fabricated via various techniques, e.g., as entire devicesor by creation of structural layers that are fused or otherwise coupled.For example, for metal FTEP devices, fabrication may include precisionmechanical machining or laser machining; for silicon FTEP devices,fabrication may include dry or wet etching; for glass FTEP devices,fabrication may include dry or wet etching, powderblasting,sandblasting, or photostructuring; and for plastic FTEP devices,fabrication may include thermoforming, injection molding, hot embossing,or laser machining. The components of the FTEP devices may bemanufactured separately and then assembled, or certain components of theFTEP devices (or even the entire FTEP device except for the electrodes)may be manufactured (e.g., using 3D printing) or molded (e.g., usinginjection molding) as a single entity, with other components added aftermolding. For example, housing and channels may be manufactured or moldedas a single entity, with the electrodes later added to form the FTEPunit (see, e.g., FIG. 16F). In some embodiments, a film or a flatsubstrate may be used to seal the bottom of the device, as shown in FIG.16B(v). The film, in some embodiments, is made from the same material asthe FTEP device, in this case, e.g., crystal styrene, cyclo-olefinpolymer (COP) or cyclic olephin co-polymers (COC). The FTEP device mayalso be formed in two or more parallel layers, e.g., a layer with thehorizontal channel and filter, a layer with the vertical channels, and alayer with the inlet and outlet ports, which are manufactured and/ormolded individually and assembled following manufacture. (See, e.g.,FIG. 13A.)

In specific aspects, the FTEP device can be manufactured using a circuitboard as a base, with the electrodes, filter and/or the flow channelformed in the desired configuration on the circuit board, and theremaining housing of the device containing, e.g., the one or more inletand outlet channels and/or the flow channel formed as a separate layerthat is then sealed onto the circuit board. The sealing of the top ofthe housing onto the circuit board provides the desired configuration ofthe different elements of the FTEP devices of the disclosure. Also, twoto many FTEP devices (up to 48 or more) may be manufactured in parallelon a single substrate, then separated from one another thereafter orused in parallel. In certain embodiments, the FTEP devices are reusableand, in some embodiments, the FTEP devices are disposable. In additionalembodiments, the FTEP devices may be autoclavable.

The electrodes 108 can be formed from any suitable metal, such ascopper, stainless steel, titanium, aluminum, brass, silver, rhodium,gold or platinum, or graphite. One preferred electrode material is alloy303 (UNS330300) austenitic stainless steel. An applied electric fieldcan destroy electrodes made from of metals like aluminum. If amultiple-use (e.g., non-disposable) flow-through FTEP device isdesired—as opposed to a disposable, one-use flow-through FTEP device—theelectrode plates can be coated with metals resistant to electrochemicalcorrosion. Conductive coatings like noble metals, e.g., gold, can beused to protect the electrode plates.

Additionally, the FTEP devices may comprise push-pull pneumatic means toallow multi-pass electroporation procedures; that is, cells toelectroporated may be “pulled” from the inlet toward the outlet for onepass of electroporation, then be “pushed” from the outlet end of theflow-through FTEP device toward the inlet end to pass between theelectrodes again for another pass of electroporation. This process maybe repeated one to many times.

Depending on the type of cells to be electroporated (e.g., bacterial,yeast, mammalian) and the configuration of the electrodes, the distancebetween the electrodes in the flow channel can vary widely. For example,in the embodiments shown in FIGS. 1A-1C, 2A-2C, 3A-3C, 4A-4E, 5A-5C, 6,and 7A-7E where the electrodes form a portion of the wall of the flowchannel where the flow channel decreases in width, the distance betweenthe electrodes in the flow channel may be between 10 μm and 5 mm, orbetween 25 μm and 3 mm, or between 50 μm and 2 mm, or between 75 μm and1 mm. In other embodiments such as those depicted in FIGS. 8A-8E, 9A-9C,10A-10E, 11A-11E, 12A-12C, 13A-13B, 14, and 16A-16D where the electrodesare positioned on either end of the channel narrowing, the distancebetween the electrodes in the flow channel may be between 1 mm and 10mm, or between 2 mm and 8 mm, or between 3 mm and 7 mm, or between 4 mmand 6 mm. The overall size of the FTEP device may be from 3 cm to 15 cmin length, or 4 cm to 12 cm in length, or 4.5 cm to 10 cm in length. Theoverall width of the FTEP device may be from 0.5 cm to 5 cm, or from0.75 cm to 3 cm, or from 1 cm to 2.5 cm, or from 1 cm to 1.5 cm.

The region of the flow channel that is narrowed is typically wide enoughso that at least two cells can fit in the narrowed portion side-by-side.For example, a typical bacterial cell is 1 μm in diameter; thus, thenarrowed portion of the flow channel of the FTEP device used totransform such bacterial cells will be at least 2 μm wide. In anotherexample, if a mammalian cell is approximately 50 μm in diameter, thenarrowed portion of the flow channel of the FTEP device used totransform such mammalian cells will be at least 100 μm wide. That is,the narrowed portion of the FTEP device will not physically contort or“squeeze” the cells being transformed.

In embodiments of the FTEP device where reservoirs are used to introducecells and exogenous material into the FTEP device, the reservoirs rangein volume from 100 μL, to 10 mL, or from 500 μL, to 75 mL, or from 1 mLto 5 mL. The flow rate in the FTEP ranges from 0.1 mL to 5 mL perminute, or from 0.5 mL to 3 mL per minute, or from 1.0 mL to 2.5 mL perminute. The pressure in the FTEP device ranges from 1-30 psi, or from2-10 psi, or from 3-5 psi.

To avoid different field intensities between the electrodes, theelectrodes should be arranged in parallel. Furthermore, the surface ofthe electrodes should be as smooth as possible without pin holes orpeaks. Electrodes having a roughness Rz of 1 to 10 μm are preferred. Inanother embodiment of the invention, the flow-through electroporationdevice comprises at least one additional electrode which applies aground potential to the FTEP device.

The electrodes are configured to deliver 1-25 Kv/cm, or 5-20 Kv/cm, or10-20 Kv/cm. The further apart the electrodes are, the more voltageneeds to be supplied; in addition, the voltage delivered of coursedepends on the types of cells being porated, the medium in which thecells are suspended, the size of the electroporation channel, and thelength and diameter of the electrodes. There are many different pulseforms that may be employed with the FTEP device, including exponentialdecay waves, square or rectangular waves, arbitrary wave forms, or aselected combination of wave forms. One type of common pulse form is theexponential decay wave, typically made by discharging a loaded capacitorto the cell sample. The exponential decay wave can be made less steep bylinking an inductor to the cell sample so that the initial peak currentcan be attenuated. When multiple waveforms in a specified sequence areused, they can be in the same direction (direct current) or differentdirections (alternating current). Using alternating current can bebeneficial in that two topical surfaces of a cell instead of just onecan be used for molecular transport, and alternating current can preventelectrolysis. The pulse generator can be controlled by a digital oranalog panel. In some embodiments, square wave forms are preferred, andin other embodiments, an initial wave spike before the square wave ispreferred.

The FTEP device may be configured to electroporate cell sample volumesbetween 1 μl to 5 ml, 10 μl to 2 ml, 25 μl to 1 ml, or 50 μl to 750 μl.The medium or buffer used to suspend the cells and material (reagent) tobe electroporated into the cells for the electroporation process may beany suitable medium or buffer for the type of cells being transformed ortransfected, such as SOC, MEM, DMEM, IMDM, RPMI, Hanks', PBS andRinger's solution, where the media may be provided in a reagentcartridge as part of a kit. Further, because the cells must be madeelectrocompetent prior to transformation or transfection, the bufferalso may comprise glycerol or sorbitol, and may also comprise asurfactant. For electroporation of most eukaryotic cells the medium orbuffer usually contains salts to maintain a proper osmotic pressure. Thesalts in the medium or buffer also render the medium conductive. Forelectroporation of very small prokaryotic cells such as bacteria,sometimes water or 10% glycerol is used as a low conductance medium toallow a very high electric field strength. In that case, the chargedmolecules to be delivered still render water-based medium moreconductive than the lipid-based cell membranes and the medium may stillbe roughly considered as conductive particularly in comparison to cellmembranes.

The compound to be electroporated into the cells can be any compoundknown in the art to be useful for electroporation, such as nucleicacids, oligonucleotides, polynucleotides, DNA, RNA, peptides, proteinsand small molecules like hormones, cytokines, chemokines, drugs, or drugprecursors.

A second aspect of the FTEP devices described herein are illustrated inFIGS. 2A-2C. FIG. 2A shows a top planar view of an FTEP device 200having an inlet 202 for introducing a fluid containing cells andexogenous material into the FTEP device 200 and an outlet 204 forremoving the transformed cells following electroporation. Cylindricalelectrodes 208 are positioned so as to define a center portion of theflow channel (not shown) where the flow channel narrows as a result ofthe curvature of the electrodes. FIG. 2B shows a cutaway view 210 fromthe top of the FTEP device 200, with the inlet 202, outlet 204, andelectrodes 208 positioned with respect to a flow channel 206. Again,note that the electrodes 208 define a narrowed portion or region of flowchannel 206. FIG. 2C shows a side cutaway view 220 of the device 200with the inlet 202 and inlet channel 212, and outlet 204 and outletchannel 214. The electrodes 208 are cylindrical and positioned in theflow channel 206 defining a narrowed portion of the flow channel 206.

A third aspect of the FTEP devices of the disclosure are illustrated inFIGS. 3A-3C. FIG. 3A shows a top planar view of an FTEP device 300having an inlet 302 for introducing a fluid containing cells andexogenous material into FTEP device 300, and an outlet 304 for removingthe transformed cells following electroporation. The semi-cylindricalelectrodes 308 are positioned so as to define a narrowed portion of aflow channel (not shown) where the channel narrows from both ends basedon the curvature of the electrodes. FIG. 3B shows a cutaway view 310from the top of the device 300, with the inlet 302, outlet 304, andelectrodes 308 positioned with respect to a flow channel 306. FIG. 3Cshows a side cutaway view 320 of the device 300 with the inlet 302 andinlet channel 312, and outlet 304 and outlet channel 314. Thesemi-cylindrical electrodes 308 are positioned in the flow channel 306so that they define a narrowed portion of the flow channel 306. Itshould be noted that the devices depicted in FIGS. 1A-1C, 2A-2C, and3A-3C show the electrodes positioned substantially mid-way along theflow channel; however, in other aspects of the devices, the electrodesmay be positioned in narrowed regions of the flow channel more towardthe inlet of the device or more toward the outlet of the device.

FIGS. 4A-4E show aspects of the FTEP devices of the disclosure withseparate inlets for the cells and the exogenous material. FIG. 4A showsa top planar view of an FTEP device 400 having a first inlet 402 forintroducing a fluid containing cells into FTEP device 400; a secondinlet 418 for introducing a fluid containing exogenous materials to beelectroporated into the cells into FTEP device 400; electrodes 408; andan outlet 404 for removing the transformed cells followingelectroporation. Although these aspects are illustrated with cylindricalelectrodes, as shown in FIG. 4A, other shaped electrodes with a curvededge—e.g., oval, semi-cylindrical, and the like as shown in relation toFIGS. 1A-1C and 3A-3C—may be used to define the flow channel asdescribed in more detail herein. FIG. 4B shows a cutaway view 410 fromthe top of the device 400, with the first inlet 402, second inlet 418,outlet 404, and electrodes 408 positioned with respect to the flowchannel 406.

FIG. 4C shows a first side cutaway view 420 of the aspect of FTEP device400 with the first inlet 402 and second inlet 418 positioned as shown inFIGS. 4A and 4B. In FIG. 4C, the first inlet channel 412 and secondinlet channel 422 meet independently with flow channel 406, and theliquid (cells and material to be porated or delivered to the cells)flows through the flow channel 406 to the outlet channel 414 and outlet404 where the transformed cells are removed from the FTEP device. Theelectrodes 408 are positioned in the flow channel 406 so that theydefine a narrowed portion of the flow channel 406. FIG. 4D shows a sidecutaway view 430 of a variation on the aspect of the FTEP device 400depicted in FIG. 4C. Here, the first inlet channel 412 and second inletchannel 424 intersect with the flow channel 406 at a three-way junction,and the liquid (cells and material to be porated or delivered to thecells) flows through the flow channel 406 to the outlet channel 414 andoutlet 404 where the transformed cells are removed from the FTEP device.The electrodes 408 are positioned in the flow channel 406 defining anarrowed portion of the flow channel 406. FIG. 4E shows a first sidecutaway view 440 of a yet another variation of the FTEP device 400 shownin FIGS. 4A and 4B. Here, the first inlet 402 and second inlet 418intersect at a junction 426 where the cells and exogenous materials mixprior to introduction of the combined fluids to the flow channel 406.The fluids flow through the flow channel 406 to the outlet channel 414and outlet 404 where the transformed cells are removed from the FTEPdevice. Electrodes 408 are positioned in the flow channel 406 so thatthey define a narrowed portion of the flow channel 406.

FIGS. 5A-5C show another aspect of the FTEP devices of the disclosurewith separate inlets for the cells and the exogenous material. FIG. 5Ashows a top planar view of an electroporation device 500 having a firstinlet 502 for introducing a fluid containing cells, a second outlet 518for introducing exogenous materials to be electroporated into the cells,and an outlet 504 for removing the transformed cells followingelectroporation. The electrodes 508 are positioned between the firstinlet 502 where the cells are introduced into the FTEP device and thesecond inlet 528 where the exogenous materials are introduced into theFTEP device. FIG. 5B shows a cutaway view 510 from the top of the FTEPdevice 500, with the first inlet 502, second inlet 528, outlet 504, andthe electrodes 508 positioned between the first inlet channel 502 andthe second inlet channel 528, where the electrodes 508 form a narrowedportion of flow channel 506. FIG. 5C shows a side cutaway view 520 ofthe FTEP device 500 with the first inlet 502 where the cells areintroduced into the FTEP device and first inlet channel 512, the secondinlet 528 where the exogenous materials are introduced into the FTEPdevice and second inlet channel 532, and an outlet channel 514 andoutlet 504 where the transformed cells are removed from the outlet 504and device. The electrodes 508 are positioned in the flow channel 506defining a narrow portion of the flow channel 506 and are positionedbetween the first inlet channel 512 and the second inlet channel 532such that the material to be introduced into the cells is added to thefluid comprising the cells after the cells have been electroporated.

FIG. 6 illustrates an FTEP device in which the flow of the fluidintroduced into the flow channel from the input channel(s) is focused,e.g., using an immiscible fluid such as an oil or a stream of air tonarrow the stream of the fluid containing the cells and the exogenousmaterials as it passes by the electrodes. FIG. 6 shows a cutaway viewfrom the top of the FTEP device 600, with the first inlet 602, secondinlet 630, outlet 604, and the electrodes 608 positioned between thefirst inlet channel 602 and outlet 604. The flow focusing is createdusing the immiscible fluid, where the electrodes 608 form a narrowedportion of flow channel 606. (For example, see, e.g., US Pub. No.2010/0184928 to Kumacheva.)

Multiplexed aspects of exemplary FTEP devices are illustrated in FIGS.7A-7E. FIG. 7A illustrates a top view of a cross section of a firstmultiplexed aspect of the FTEP devices of the disclosure. The FTEPdevice in FIG. 7A is a multiplexed FTEP device 700 in which parallelflow channels 706 for each FTEP unit are defined in part by sharedcylindrical electrodes 708 a-708 f forming devices (i), (ii), (iii),(iv), and (v). Each flow channel 706 has an inlet 702 for introducingdifferent sets of cells and/or exogenous materials into the FTEP unitsand an outlet 704 for removing the transformed cells from the FTEPunits. Adjacent units share electrodes, where the electrodes alternatecharge, e.g., +/−/+/−/+(that is, if electrode 708 a is +, electrode 708b is −, electrode 708 c is +, electrode 708 d is −, and so on). FIG. 7Bis an illustration of a top view of a cross section of a secondmultiplexed aspect of the FTEP devices 710 of the disclosure. This is amultiplexed device 710 in which parallel flow channels 706 are definedin part by shared oval electrodes 708 a-708 f. Each flow channel 706 hasan inlet 702 for introducing different sets of cells and/or exogenousmaterials into the flow channels 706, and an outlet for removing thetransformed cells from FTEP units (i), (ii), (iii), (iv), and (v).Again, adjacent devices share electrodes, where the electrodes alternatecharge, e.g., +/−/+/−/+.

FIG. 7C is an illustration of a top view of a cross section of a thirdmultiplexed aspect of the FTEP devices of the disclosure. In thisexemplary multiplexed FTEP device 720, the individual FTEP units arestaggered. The parallel flow channels 706 are defined in part byindividual cylindrical electrodes 714 a-714 j that are not shared asshown in FIGS. 7A and 7B. Each flow channel 706 has its own pair ofelectrodes 708, an inlet 702 for introducing different sets of cellsand/or exogenous materials into the FTEP device, and an outlet forremoving transformed cells from the FTEP units (i), (ii), (iii), (iv),and (v). FIG. 7D is an illustration of a top view of a cross section ofanother exemplary multiplexed FTEP device. In this multiplexed FTEPdevice 730, staggered, parallel flow channels 706 are defined in part byindividual oval electrodes 716 a-716 j. Each flow channel 706 has itsown un-shared pair of electrodes 716 (e.g., 716 a/716 b, 716 c/716 d,716 e/716 f, 716 g/716 h, and 716 i/716 j), an inlet 702 for introducingdifferent sets of cells and/or exogenous materials into the FTEP units,and an outlet 704 for removing transformed cells from the FTEP units.FIG. 7E is an illustration of a top view of a cross section of anotherexemplary multiplexed FTEP device. In this exemplary multiplexed device740, staggered, parallel flow channels 706 are defined in part byindividual half-cylindrical electrodes 716 a-716 j. Each flow channel706 has its own pair of electrodes 716, a separate inlet 702 forintroducing different sets of cells and/or exogenous materials into theFTEP unit, and an outlet 704 for removing the transformed cells from theFTEP unit.

Additional aspects of the FTEP devices of the disclosure are illustratedin FIGS. 8A-8E. Note that in the FTEP devices in FIGS. 8A-8E (and inFIGS. 9-14), the electrodes are not positioned on either side of theflow channel to narrow the flow channel; instead, the electrodes areplaced such that a first electrode is placed between the inlet and thenarrowed region of the flow channel, and the second electrode is placedbetween the narrowed region of the flow channel and the outlet. FIG. 8Ashows a top planar view of an FTEP device 800 having an inlet 802 forintroducing a fluid containing cells and exogenous material into FTEPdevice 800 and an outlet 804 for removing the transformed cells from theFTEP following electroporation. The electrodes 808 are introducedthrough channels (not shown) in the device. FIG. 8B shows a cutaway view810 from the top of the device 800, with the inlet 802, outlet 804, andelectrodes 808 positioned with respect to a flow channel 806. FIG. 8Cshows a side cutaway view 820 of the device 800 with the inlet 802 andinlet channel 812, and outlet 804 and outlet channel 814. The electrodes808 are positioned in electrode channels 816 so that they are in fluidcommunication with the flow channel 806, but not directly in the path ofthe cells traveling through the flow channel 806. Again, note that thefirst electrode is placed between the inlet and the narrowed region ofthe flow channel, and the second electrode is placed between thenarrowed region of the flow channel and the outlet

An expanded side cutaway view 830 of the bottom portion of the device800 in FIG. 8D shows that the electrodes 808 in this first aspect of thedevice 830 are positioned in the electrode channels 816 which aregenerally perpendicular to the flow channel 806 such that the fluidcontaining the cells and exogenous material flows from the inlet channel812 through the flow channel 806 to the outlet channel 814, and in theprocess fluid flows into the electrode channels 816 to be in contactwith the electrodes 808. In this aspect, the inlet channel, outletchannel and electrode channels all originate from the same planar sideof the device, as shown in FIGS. 8C and 8D. In certain aspects, however,such as that shown in FIG. 8E, the electrodes are introduced from adifferent planar side of the FTEP device than the inlet and outletchannels. Here, the electrodes 808 in this alternative aspect 840 of thedevice 800 are positioned in the electrode channels 816 perpendicular tothe flow channel 806 such that fluid containing the cells and exogenousmaterial flows from the inlet channel 812 through the flow channel 806to the outlet channel 814. The cells and exogenous material in bufferflow into the electrode channels 816 to be in contact with bothelectrodes 808. In this aspect, the inlet channel and outlet channeloriginate from a different planar side of the device than do theelectrodes and electrode channels.

FIGS. 9A-9C illustrate yet another aspect of the FTEP devices of thedisclosure. FIG. 9A shows a top planar view of an FTEP device 900 havinga first inlet 902 for introducing a fluid containing cells into FTEPdevice 900 and an outlet 904 for removing the transformed cells from theFTEP device following electroporation. However, in this FTEP device,there is a second inlet 922 for introducing exogenous material to beelectroporated to the cells. The electrodes 908 are introduced throughchannels (not shown) machined into the device. FIG. 9B shows a cutawayview 910 from the top of the FTEP device 900, with the first inlet 902,second inlet 922, outlet 904, and the electrodes 908 positioned withrespect to the flow channel 906. FIG. 9C shows a side cutaway view 920of the device 900 with the inlet 902 and inlet channel 912, and outlet904 and outlet channel 914. The electrodes 908 are positioned in theelectrode channels 916 so that they are in fluid communication with theflow channel 906, but not substantially in the path of the cellstraveling through the flow channel 906. The electrodes 908 in thisaspect of the FTEP device 920 are positioned in the electrode channels916 where the electrode channels 916 are generally perpendicular to theflow channel 906 such that fluid containing the cells and fluidcontaining the exogenous materials flow from the inlets 902, 922 throughthe inlet channels 912, 924 into the flow channel 906 and through to theoutlet channel 914, and in the process the cells and exogenous materialin medium flows into the electrode channels 916 to be in contact withthe electrodes 908. One of the two electrodes 908 and electrode channels916 is positioned between inlets 902 and 922 and inlet channels 912 and924 and the narrowed region (not shown) of flow channel 906, and theother electrode 908 and electrode channel 916 is positioned between thenarrowed region (not shown) of flow channel 906 and the outlet channel914 and outlet 904. In FIG. 9C, the inlet channel, outlet channel andelectrode channels all originate from the same planar side of thedevice, although the electrodes (and inlets and outlet) can also beconfigured to originate from different planar sides of the FTEP devicesuch as illustrated in FIG. 8E.

FIGS. 10A-10E illustrate yet another aspect of the devices of thedisclosure. FIG. 10A shows a top planar view of an electroporationdevice 1000 having an inlet 1002 for introducing a fluid containingcells and exogenous material into the FTEP device 1000 and an outlet1004 for removal of the transformed cells from the FTEP device 1000following electroporation. The electrodes 1008 are introduced throughchannels (not shown) machined into the device. FIG. 10B shows a cutawayview 1010 from the top of the device 1000, showing an inlet 1002, anoutlet 1004, a filter of substantially uniform density 1050, andelectrodes 1008 positioned with respect to the flow channel 1006. FIG.10C shows a cutaway view 1020 from the top of an alternativeconfiguration of the device 1000, with an inlet 1002, an outlet 1004, afilter of substantially increasing gradient density 1050, and electrodes1008 positioned with respect to the flow channel 1006. In FIGS. 10A-E,like FIGS. 9A-9C, the first electrode is placed between the inlet andthe narrowed region of the flow channel, and the second electrode isplaced between the narrowed region of the flow channel and the outlet.In some embodiments such as those depicted in FIGS. 10A-10E, the FTEPdevices comprise a filter disposed within the flow channel positioned inthe flow channel after the inlet channel and before the first electrodechannel. The filter may be substantially homogeneous in porosity (e.g.,have a uniform density as in FIG. 10B); alternatively, the filter mayincrease in gradient density where the end of the filter proximal to theinlet is less dense, and the end of the filter proximal to the narrowedportion of the flow channel has greater gradient density (as shown inFIG. 10C). The filter may be fashioned from any suitable and preferablyinexpensive material, including porous plastics, hydrophobicpolyethylene, cotton, glass fibers, or the filter may be integral withand fabricated as part of the FTEP device body (see, e.g., FIG. 15E).

FIG. 10D shows a side cutaway view 1030 of the device 1000 with an inlet1002 and an inlet channel 1012, and an outlet 1004 and an outlet channel1014. The electrodes 1008 are positioned in the electrode channels 1016so that they are in fluid communication with the flow channel 1006, butnot directly in flow channel 1006. Note that filter 1050 is positionedbetween inlet 1002 and inlet channel 1012 and electrodes 1008 andelectrode channels 1016. An expanded side cutaway view 1040 of thebottom portion of the FTEP device 1000 in FIG. 10E shows that theelectrodes 1008 in this aspect of the FTEP device 1000 are positioned inthe electrode channels 1016 and perpendicular to the flow channel 1006such that fluid containing the cells and exogenous material flows fromthe inlet channel 1012 through the flow channel 1006 to the outletchannel 1014, and in the process fluid flows into the electrode channels1016 to be in contact with both electrodes 1008. In FIGS. 10D and 10E,the inlet channel, outlet channel and electrode channels all originatefrom the same planar side of the device, although the electrodes (andthe inlets and outlet) can also be configured to originate from adifferent planar side such as illustrated in FIG. 8E.

FIGS. 11A-11E illustrate other aspects of the FTEP devices of thedisclosure. FIG. 11A shows a top view of an FTEP device 1100 having afirst inlet 1102 for introducing a fluid containing cells into the FTEPdevice and a second inlet 1122 for introducing a fluid containingexogenous materials to be introduced to the cells into the FTEP device,electrodes 1108 positioned in electrode channels (not shown), and anoutlet 1104 for removal of the transformed cells followingelectroporation. FIG. 11B shows a cutaway view 1110 from the top of thedevice 1100, comprising a first inlet 1102, second inlet 1122, outlet1104, filter 1150, and electrodes 1108 positioned with respect to theflow channel 1106. Again, note that the electrodes 1108 are positionedso that the first electrode is on the “inlet end” of the narrowed regionin flow channel 1106 and the second electrode is on the “outlet end” ofthe narrowed region in flow channel 1106. FIG. 11C shows a first sidecutaway view 1120 of a fifth aspect of the device 1100 with the firstinlet 1102 and second inlet 1122 positioned as shown in FIG. 11A. Thefirst inlet channel 1112 and second inlet channel 1124 meet separatelywith the flow channel 1106 prior to encountering filter 1150, and theliquid flows from the inlet channels 1112 and 1124 through the flowchannel 1106 (and filter 1150) to the outlet channel 1114 and outlet1104. The electrodes 1108 are positioned in the electrode channels 1116so that they are in fluid communication with the flow channel 1106, butnot directly in flow channel 1106. Note that in some embodiments,electrodes 1108 may be positioned in electrode channels 1116 such thatelectrodes 1108 are flush with the walls of flow channel 1106 (e.g., seeFIG. 15F(iii)). Alternatively, electrodes 1108 may extend a minimaldistance into flow channel 1106; however, in doing so electrodes 1108 donot extend into flow channel 1106 to the extent that the electrodesimpede the flow of the cells through the flow channel.

FIG. 11D shows a side cutaway view 1130 of a variation of the aspect ofthe FTEP device 1100 shown in FIGS. 11A-11C with the first inlet 1102and second inlet 1122 positioned as shown in FIG. 11A. The first inletchannel 1112 and second inlet channel 1124 intersect with flow channel1106 at a three-way junction with flow channel 1106 and prior toencountering filter 1150. The liquid flows through the flow channel 1106to the outlet channel 1114 and outlet 1104. The electrodes 1108 arepositioned in the electrode channels 1116 so that they are in fluidcommunication with the flow channel 1106, but not directly in the flowchannel 1106. Again, the electrodes 1108 are positioned so that thefirst electrode is on the “inlet end” of the narrowed region in flowchannel 1106 and the second electrode is on the “outlet end” of thenarrowed region in flow channel 1106. FIG. 11E shows a side cutaway view1140 of yet another variation on the aspect of the FTEP device 1100shown in FIGS. 11A-11C. The first inlet channel 1112 and second inletchannel 1126 intersect at a junction into a single channel prior tointersecting flow channel 1106. The fluids flow from the inlets 1102 and1122, through the inlet channels 1112 and 1126, into and through flowchannel 1106 and the filter 1150, into electrode channels 1116 (suchthat electrodes 1108 are in fluid communication with flow channel 1106)and continuing through flow channel 1106 to the outlet channel 1114 andfinally to the outlet 1104 where the transformed cells are removed fromthe FTEP device 1100. The electrodes 1108 are positioned in theelectrode channels 1116 so that they are in fluid communication with theflow channel 1106, but not directly in the flow path of the cellstraveling through the flow channel 1106. Although each of FIGS. 11C-11Eshow the inlet channels, outlet channel and electrode channelsoriginating from the same planar side of the device, all of the inlets,outlet and electrodes in each of these aspects can also be configured tooriginate from different planar sides of the FTEP device.

FIGS. 12A-12C illustrate another aspect of the FTEP devices of thedisclosure. FIG. 12A shows a top view of an electroporation device 1200having a first inlet 1202 for introducing a fluid containing cells intoFTEP device 1200, a second inlet 1228 for introducing exogenousmaterials to be porated into the cells into FTEP device 1200, and anoutlet 1204 for removing transformed cells from FTEP device 1200following electroporation. The electrodes 1208 are introduced throughchannels (not shown) machined into the device and are positioned betweenthe first inlet 1202 and the second inlet 1228. FIG. 12B shows a cutawayview 1210 from the top of the device 1200, with the first inlet 1202,second inlet 1228, outlet 1204, and the electrodes 1208 positioned withrespect to the flow channel 1206. Additionally, the FTEP device depictedin FIG. 12B comprises a filter disposed between the first inlet 1202 andthe first electrode 1208 and before the narrowed region of flow channel1206. Filter 1250 in this embodiment has a gradient of pore sizes, fromlarge to small. FIG. 12C shows a side cutaway view 1220 of FTEP device1200 comprising a first inlet 1202 and first inlet channel 1212, afilter 1250, a second inlet 1228 and second inlet channel 1232, and anoutlet 1204 and outlet channel 1214. The electrodes 1208 are positionedin the electrode channels 1216 perpendicular to flow channel 1206 andbetween the first and second inlets. The electrodes 1208 are in fluidcommunication with flow channel 1206, but not substantially in the flowpath of the cells traveling through flow channel 1206. Exogenousmaterials are added to FTEP device 1200 via the second inlet 1228 andthrough the second inlet channel 1232 and encounter the cells after thecells are electroporated. In FIG. 12C, the inlet channels, outletchannel and electrode channels all originate from the same planar sideof the device, although these features can also be configured tooriginate from different planar sides of FTEP device 1200.

FIGS. 13A and 13B show the side and top cutaway views, respectively, ofyet another aspect of the invention. FIG. 13A shows a multilayer device1300 with a top layer 1352 having an inlet 1302 and an inlet channel1312, a flow channel 1306, and outlet 1304 and an outlet channel 1314.The electrodes 1308 are on bottom layer 1356, e.g., provided as stripson a solid substrate. The middle layer 1354 is a solid substrate withelectrode channels 1316 provided therein, and the electrode channels1316 in this aspect provide fluid communication between the electrodes1308 of bottom layer 1356 and flow channel 1306 of top layer 1352. Thecells and exogenous materials in fluid are introduced to the FTEP device1300 via inlet 1302 and flow through inlet channel 1312 and into flowchannel 1306, and then to the outlet channel 1314. In the process, thefluid flows into electrode channels 1316 so that electrodes 1308 are influid contact with flow channel 1306. The cells are porated as they passthrough flow channel 1306 between the two electrodes 1308. FIG. 13Bshows the top view of a cutaway 1310 of this aspect of the FTEP device1300 showing the position of the inlet 1302, outlet 1304, electrodes1308 and electrode channels 1316 with respect to the flow channel 1306.Although the electrodes are shown here as strips, they may also beconfigured to be other shapes, e.g., round, cylindrical, asymmetric,rectangular, or square.

FIG. 14 illustrates an FTEP device in which flow focusing 1430 of thefluid introduced into the flow channel from the input channel(s) takesplace, e.g., using an immiscible fluid such as an oil or using air tofocus (narrow) the stream of the fluid containing the cells andexogenous materials as the fluid encounters the electrode channels, andthe electrodes. FIG. 14 shows a cutaway view from the top of the device1400, with the first inlet 1402, the flow focusing of the fluid after itexits the inlet channel and enters the flow channel 1406, and theelectrodes 1408 positioned between the inlet 1402 and the outlet 1404,where the electrodes 1408 are positioned on either end of a narrowedportion of flow channel 1406.

FIGS. 15A through 15C are top perspective, bottom perspective, andbottom views, respectively, of six co-joined FTEP devices 1550 that maybe part of, e.g., reagent cartridge 1600 in FIGS. 16A and 16B infra(i.e., serve as FTEP 1606 in reagent cartridge 1600). FIG. 15A depictssix FTEP units 1550 arranged on a single, integrally-formed injectionmolded cyclic olefin copolymer (COC) substrate 1556. The channels 1506shown in FIG. 15B are sealed with a COC film having a thickness of 50microns to 1 mm (not shown). The COC film may be thermally bonded to thebase of the assembly 1500 (the surface most prominently displayed inFIG. 15B). In FIGS. 15B and 15C, the co-joined FTEP devices havedifferent channel architectures and electrode placements that may beadvantageous in various applications. For instance, the curved channelsof devices (i), (iv) and (v) take advantage of inertia to direct thecells in the fluid away from the electrodes. The electrodes may bepositioned off center in the channel to further enhance cells flow andreduce the potential for damage to the cells. This may be particularlyimportant for cells or materials that are particularly sensitive toelectrolytic effects or local changes in pH proximate the electrodes.The electrodes may be at least partially embedded into the channelwalls, as shown in embodiments (iii) and (iv), so as to further reducethese effects.

Each of the six FTEP units 1550 have wells 1552 that define cell sampleinlets and wells 1554 that define cell sample outlets. FIG. 15B is abottom perspective view of the six co-joined FTEP devices 1550 of FIG.15A also depicting six FTEP units 1550 arranged on a single substrate1556. Six inlet wells 1552 can be seen, one for each flow-throughelectroporation unit 1550, and one outlet well 1554 can be seen. Alsoseen in FIG. 15B for each FTEP unit 1550 are an inlet 1502, an outlet1504, a flow channel 1506, and two electrodes 1508 on either end of anarrowed region in flow channel 1506. Filters 1570 and 1572 are includedin the channels to prevent clogging of the channel, particularly atnarrowed region of the flow channel. FIG. 15C is a bottom view of thesix co-joined FTEP devices 1550 of FIGS. 15A and 15B. Depicted in FIG.15C are six FTEP units 1550 arranged on a single substrate 1556, whereeach FTEP unit 1550 comprises an inlet 1502, outlet 1504, flow channel1506 and two electrodes 1508 on either end of a narrowed region in flowchannel 1506 in each FTEP unit 1550. Once the six FTEP units 1550 arefabricated, they can be separated from one another (e.g., “snappedapart”) upon the depicted score lines and used one at a time as seen inthe cartridge depicted in FIG. 16A or 16B; alternatively, the FTEP unitsmay be used in embodiments where two or more FTEP units 1550 are used inparallel.

FIG. 15D shows scanning electromicrographs of the FTEP units depicted inFIG. 15C with the units (i), (ii), (iii), (iv), (v), and (vi) in FIG.15D corresponding to units (i), (ii), (iii), (iv), (v), and (vi) in FIG.15C. In FIG. 15D, for each unit both the electrode channels 1516 as wellas the flow channel 1506 can be seen. The scale is 1 mm per hash mark asshown in the lower right-hand corner of each micrograph. It can be seenthat in this embodiment the inlet apertures have a rounded edge, theadvantages of which include resistance to air trapping, promotion oflaminar flow, and reduction of risk of cell damage. The rounded edgesmay have a radius of curvature of about 10, 20, 30, 40, 50, 60, 70, 80,90, 100, 150, 200 or 250 microns.

FIG. 15E shows scanning electromicrographs of the filters 1570 and 1572depicted as black bars in FIGS. 15B and 15C. Note in this embodiment,the porosity of the filter 1572 varies from large pores (near the inlet1502) to small pores toward the flow channel (not shown). In thisembodiment, the channel optionally but not necessarily narrows. If asecond filter is present, the second filter may also vary in porosity.In the case of a second filter between the second electrode and theoutlet channel, the filter can vary from large pores (near the secondelectrode) to small pores toward the outlet channel. Scale informationis shown in each micrograph.

In certain embodiments, the filter serves the purpose of filtering thefluid containing the cells and DNA before the fluid encounters thenarrowed portion of the flow channel. The filter thus decreases thelikelihood that cells or other matter do not clog the narrowed portionof the flow channel. Instead, if there is particulate matter that posesa threat to clogging the narrowed portion of the flow channel, thefilter will catch the particulate matter leaving other pores throughwhich the rest of the cell/DNA/fluid can move. The depicted construction(integral molding with the channel wall) is particularly advantageousbecause it reduces cost and complexity of the device while also reducingthe risk that pieces of the filter itself may dislodge and clog thechannel or otherwise interfere with device operation. Note that in thisembodiment, the filter has a gradient pore size (from large poresproximate the inlet to smaller pores proximal the narrowed portion ofthe flow channel); however, in alternative embodiments the pores may bethe same size or not gradient in size.

Further, in yet other embodiments, the flow channel may not narrow. Inthese specific embodiments, the pores themselves can serve to providesuch a narrowing function for enhancing electroporation, and the flowchannel walls do not constrict or constrict minimally as the fluid flowsthrough the channel. These embodiments can allow control of the rate offlow of cells through the device to optimize introduction of exogenousmaterial into various cell types.

Moreover, though the scanning electromicrographs in FIG. 15E shown thefilter elements as rounded “pegs”, it should be understood that thefilter elements may be triangular-, square-, rectangular-, pentagonal-,hexagonal-, oval-, elliptical- or other faceted-shaped pegs.

FIG. 15F depicts (i) the electrodes 1508 before insertion into the FTEPdevice 1500 (here, a six-unit FTEP device) having inlet reservoirs 1552and outlet reservoirs 1554. In the preferred embodiment, the device 1500is used in an orientation inverted relative to that shown in FIG. 15F(i). FIG. 15F (ii) depicts an electrode 1508 contained within andprojecting from a sheath. FIG. 15F (iii) depicts the electrode 1508inserted into an electrode channel 1516 with the electrode channel 1516(and electrode 15080 adjacent to the flow channel 1506. In theembodiment shown in FIG. 15F (iii), the electrode is even with the wallsof the flow channel; that is, the electrode is not in the path of thecells/DNA/fluid flowing through flow channel 1506, however, neither isthe electrode recessed within the electrode channel 1516. Indeed, theelectrode 1508 may be recessed within the electrode channel 1516, may beextend to the end of electrode channel 1516 and thus be even with thewalls of flow channel 1506, or electrode 1508 may extend a minimaldistance into flow channel 1506 so long as the electrode does not impedemovement of the cells through the flow channel. The rounded or bevelededges of the aperture in the flow channel 1506 help prevent trapping airand reduce discontinuities in the electric field.

FIG. 15G presents two scanning electromicrographs of two differentconfigurations of the aperture where electrode channel 1516 meets flowchannel 1506. In FIG. 15G (i) (top), the edge of the junction ofelectrode channel 1516 and flow channel 1506 comprises a sharp edge. Incontrast, in FIG. 15G (ii) (bottom), the edges of the junction ofelectrode channel 1516 and flow channel 1506 comprises a rounded edge.Both configurations were tested (data not shown), and it was found thatthe rounded-edge configuration decreases the likelihood that air willbecome trapped between flow channel 1506 and the electrode (not seen inthis Figure) in electrode channel 1516. Indeed, the electrodes of theFTEP devices should be “wet”; that is, immersed in the fluid/cells/DNA.

Automated Multi-Module Cell Processing System(s) Comprising the FTEPs

FIG. 16A depicts an exemplary combination reagent cartridge and FTEPdevice 1600 (“cartridge”) that may be used in an automated multi-modulecell processing system. Cartridge 1600 comprises a body 1602, andreagent receptacles or reservoirs 1604. Additionally, cartridge 1600comprises an FTEP device 1606, aspects of which are described inrelation to FIGS. 1-6 and 8-15 (e.g., in this embodiment of thecartridge, there is a single FTEP device). Cartridge 1600 may bedisposable or cartridge 1600 may be configured to be reused. Preferably,cartridge 1600 is disposable. Cartridge 1600 may be made from a varietyof suitable materials, including stainless steel, aluminum, or plasticsincluding polyvinyl chloride, polyethylene, polyamide, polyethylene,polypropylene, acrylonitrile butadiene, polycarbonate,polyetheretheketone (PEEK), poly(methyl methylacrylate) (PMMA),polysulfone, and polyurethane, and co-polymers of these and otherpolymers. If the cartridge is disposable, preferably it is made ofplastic. Preferably the material used to fabricate the cartridge isthermally-conductive, as in certain embodiments the cartridge 1600contacts a thermal device (not shown) that heats or cools reagents inthe reagent receptacles or reservoirs 1604. In some embodiments, thethermal device is a Peltier device or thermoelectric cooler. Reagentreceptacles or reservoirs 1604 may be receptacles into which individualtubes of reagents are inserted as shown in FIG. 16A, receptacles intowhich one or more multiple co-joined tubes are inserted (e.g., rows offive tubes that are co-joined are inserted into the reagent receptaclesas seen in FIG. 16B (iv)), or the reagent receptacles may hold thereagents without inserted tubes. Additionally, the receptacles in areagent cartridge may be configured for any combination of tubes,co-joined tubes, and direct-fill of reagents.

In one embodiment, the reagent receptacles or reservoirs 1604 of reagentcartridge 1600 are configured to hold various size tubes, including,e.g., 250 ml tubes, 25 ml tubes, 10 ml tubes, 5 ml tubes, and Eppendorfor microcentrifuge tubes. In yet another embodiment, all receptacles maybe configured to hold the same size tube, e.g., 5 ml tubes, andreservoir inserts may be used to accommodate smaller tubes in thereagent reservoir. In yet another embodiment—particularly in anembodiment where the reagent cartridge is disposable—the reagentreservoirs hold reagents without inserted tubes. In this disposableembodiment, the reagent cartridge may be part of a kit, where thereagent cartridge is pre-filled with reagents and the receptacles orreservoirs sealed with, e.g., foil, heat seal acrylic or the like andpresented to a consumer where the reagent cartridge can then be used inan automated multi-module cell processing system. The reagents containedin the reagent cartridge will vary depending on work flow; that is, thereagents will vary depending on the processes to which the cells aresubjected in the automated multi-module cell processing system.

Further, FIG. 16A shows additional detail for an embodiment of a reagentcartridge and FTEP device where reagent receptacles or reservoirs 1604are configured to accept tubes 1660 or thermal spacers 1662. Inaddition, within thermal spacer 1662 is a small tube 1654, such as anEppendorf or microcentrifuge tube, useful when only small reagentvolumes are required. Thermal spacer 1662 is thermally conductiveassuring heat or cooling is transferred to reagents contained in smalltubes, e.g., Eppendorf tubes 1654. As discussed above, in someembodiments the body of the reagent cartridge itself is thermallyconductive and is in contact with a thermal device to warm or cool thereagents contain therein as desired by a user. Also seen in FIG. 16A arefoil seals 1658 used to seal tubes 1660 and 1654. Alternatively, if thereagent cartridge is reusable, the reagent cartridge may comprise athermal device to heat and cool reagents contained within, as opposed tocontacting a thermal device.

In certain embodiments of reagent cartridge 1600 shown in FIG. 16A, thereagent cartridge comprises an optically readable code (e.g., barcode orAztec code) or instructions/data stored in an onboard memory element(not shown) readable by complementary sensor of the automated system(see applications incorporated by reference) and transmitted to aprocessor of the automated system. The code, data, instructions, orscript provide (or enable the retrieval of) instructions for dispensingby the automated system the reagents and controlling the electroporationdevice contained within reagent cartridge 1600. Also, the reagentcartridge 1600 as one component in an automated multi-module cellprocessing system may include a code, instructions or scripts specifyingtwo, three, four, five, ten or more processes performed by the automatedmulti-module cell processing system, or even specify all processesperformed by the automated multi-module cell processing system. Incertain embodiments, the reagent cartridge is disposable and ispre-packaged with reagents tailored to performing specific cellprocessing protocols, e.g., genome editing or protein production.Because the reagent cartridge contents vary while components of theautomated multi-module cell processing system may not, the scriptassociated with a particular reagent cartridge matches the reagents usedand cell processes performed. Thus, e.g., reagent cartridges may bepre-packaged with reagents for genome editing and a script thatspecifies the process steps for performing genome editing in anautomated multi-module cell processing system.

FIG. 16B depicts an alternative embodiment of a combination reagentcartridge and electroporation device. At 16B (i), a body 1602 of areagent cartridge is shown, as are reagent receptacles or reservoirs1604, that may be configured to hold various size tubes, including,e.g., 250 ml tubes, 25 ml tubes, 10 ml tubes, 5 ml tubes, and Eppendorfor microcentrifuge tubes. Additionally, there is a recess 1610 intowhich an FTEP device (not shown) may be placed. FIG. 16B (ii) depicts acover 1652 for the body 1602 of the reagent cartridge. FIG. 16B (iii)depicts the body 1602 and cover 1652 of the FTEP device assembled, withan assembled FTEP device 1660 placed within recess 1610 (seen in (i)).FIG. 16B (iv) shows co-joined tubes that may be placed within reagentreceptacles or reservoirs 1604. FIG. 16B (v) is an exploded view of theFTEP cartridge 1606, covers 1608 with seals or gaskets (not shown) thatmate with the inlet and outlet reservoirs, and film 1610, which is usedto seal the bottom of the FTEP device. The covers and seals form anair-tight seal permitting the application of pneumatic pressuresufficient to drive the fluids in the FTEP device in the mannerdescribed above. Element 1610 corresponds to the COC film describedabove in connection with FIG. 15 which seals the channels in theunderside of the cartridge 1606 and serves as the base or bottom of theFTEP device 1600.

FIG. 17 depicts an exemplary automated multi-module cell processinginstrument 1700 comprising an exemplary FTEP device 1730 as part of areagent cartridge 1710 to, e.g., perform one of the exemplary workflowsdescribed below, as well as additional exemplary workflows. Illustratedis a gantry 1702, providing an automated mechanical motion system(actuator) (not shown) that supplies XYZ axis motion control to, e.g.,modules of the automated multi-module cell processing instrument 1700,including, e.g., an air displacement pipette 1732. In some automatedmulti-module cell processing instruments, the air displacement pipettoris moved by a gantry and the various modules and reagent cartridgesremain stationary; however, in other embodiments, the pipetting systemmay stay stationary while the various modules are moved. Also includedin the automated multi-module cell processing instrument 1700 is wash orreagent cartridge 1704, comprising reservoirs 1706. Wash or reagentcartridge 1704 may be configured to accommodate large tubes, forexample, wash solutions, or solutions that are used often throughout aniterative process. In one example, wash or reagent cartridge 1704 may beconfigured to remain in place when two or more reagent cartridges 1710are sequentially used and replaced. Although reagent cartridge 1710 andwash or reagent cartridge 1704 are shown in FIG. 17 as separatecartridges, the contents of wash cartridge 304 may be incorporated intoreagent cartridge 1710.

The exemplary automated multi-module cell processing instrument 1700 ofFIG. 17 further comprises a cell growth module 1734. In the embodimentillustrated in FIG. 17, the cell growth module 1734 comprises two cellgrowth units 1718, 1720 as well as a cell concentration module 1722. Inalternative embodiments, the cell concentration module 1722 may beseparate from cell growth module 1734, e.g., in a separate, dedicatedmodule. Also illustrated as part of the automated multi-module cellprocessing instrument 1700 of FIG. 17 is screening/selection module1728, served by, e.g., air displacement pipettor 1732, and filtrationmodule 1724. Also seen are a waste repository 1726, and a nucleic acidassembly/desalting module 1714 comprising a reaction chamber or tubereceptacle (not shown) and further comprising a magnet 1716 to allow forpurification of nucleic acids using, e.g., magnetic solid phasereversible immobilization (SPRI) beads (Applied Biological MaterialsInc., Richmond, BC). The reagent cartridge, transformation module, andcell growth module are described in greater detail below.

FIG. 18 is a block diagram of one embodiment of a method 1800 for usingthe automated multi-module cell processing system depicted in FIG. 17.In a first step, cells are transferred 1801 from reagent cartridge 1810to growth vial 1818. The cells are incubated 1802, e.g., until they growto a desired OD 1803. The cells are then transferred 1804 to filtrationmodule 1822 to render the cells electrocompetent and to reduce thevolume of the cell sample to a volume appropriate for electroporation,as well as to remove unwanted components, e.g., salts, from the cellsample. Once the cells have been rendered electrocompetent and suspendedin an appropriate volume for transformation, the cell sample istransferred 1812 to FTEP device 1830 in reagent cartridge 1810.

While cells are being processed for electroporation, automatedmulti-module cell processing system may be preparing the nucleic acidsto be electroporated into the cells. As a first step, a nucleic acidsample comprising vectors, a nucleic acid sample comprisingoligonucleotides, and enzymes and other reaction components aretransferred 1806 from reagent reservoirs of the reagent cartridge to adisposable tube in the nucleic acid assembly module, and the nucleicacid assembly mix (vectors+oligos+enzymes+reagents) is incubated 1807.Once sufficient time has elapsed for the nucleic acid assembly reactionto take place, the nucleic acid assembly mix is combined with magneticbeads 1808. The mix is incubated for sufficient time for the assembledvector and oligonucleotides to bind to the magnetic beads. The magnet isengaged 1809 so that the assembled vector and oligonucleotides can bewashed 1810 and eluted 1811. Once the assembled vector has been eluted1811, the assembled vector is transferred 1812 to the FTEP device in thereagent cartridge. The assembled vector and the cells are thus combinedin an FTEP device and the FTEP device is engaged 1813.

After electroporation, the transformed cells optionally are transferred1814 to a second growth vial to, e.g., recover from the transformationprocess, be submitted to selection, or for, in this particular example,genome editing. Once the transformed cells have recovered, been selected(e.g., by an antibiotic or other reagent added from the reagentcartridge or by, e.g., temperature), or genome editing has taken place,the cells may be removed from the instrument and used in furtherresearch 1818, or aspirated 1815 by the filtration module to be washedto remove dead cells and/or concentrated, rendered electrocompetent, andeluted 1816 using a wash solution dispensed through the filter fromreagent reservoir in wash or reagent cartridge. The eluted cells maythen be collected in an empty vessel in the wash cartridge. The airdisplacement pipettor may transfer media from the reagent cartridge tothe eluted cells. All or some of steps 1801-1816 may be repeated forrecursive rounds of genome editing 1817; alternatively, the transformedcells may be used in research 1818.

Use of the Reagent Cartridge(s) in Exemplary Automated Multi-Module CellProcessing Systems

As described above, the FTEP devices may be used in stand-alone devicesor used as a module in an automated multi-module processing system. Ageneral exemplary embodiment of a multi-module cell processing system isshown in FIG. 19. In some embodiments, the cell processing system 1900may include a housing 1960, a receptacle for introducing cells to betransformed or transfected 1902, and a growth module (a cell growthdevice) 1904. The cells to be transformed are transferred from a reagentcartridge to the growth module to be cultured until the cells hit atarget OD. Once the cells hit the target OD, the growth module may coolor freeze the cells for later processing or transfer the cells to afiltration module 1920 where the cells are rendered electrocompetent andconcentrated. The filtration module 1920 comprises e.g., a filter totreat the cells to make them electrocompetent and concentrate theelectrocompetent cells. In one example, 20 ml of cells+growth media isconcentrated to 400 μl cells in 10% glycerol. Once the electrocompetentcells have been concentrated, the cells are transferred to anelectroporation device in the reagent cartridge to be transformed with adesired nucleic acid. In addition to the receptacle for receiving cells,the multi-module cell processing system includes a receptacle located inthe reagent cartridge for storing the nucleic acids to be electroporatedinto the cells 1906. The nucleic acids are transferred to theelectroporation device 1908 which already contains the concentratedelectrocompetent cells grown to the specified OD, where the nucleicacids are introduced into the cells. Following electroporation, thetransformed cells are transferred into, e.g., a recovery module 1910.Here, the cells are given the opportunity to recover from theelectroporation procedure.

In some embodiments, after recovery the cells are transferred to astorage module 1912 to be stored at, e.g., 4° C. or frozen. The cellscan then be retrieved from a retrieval module 1914 and used for furtherstudies off-line. The automated multi-module cell processing system iscontrolled by a processor 1950 configured to operate the instrumentbased on user input or one or more scripts, where one or more may beassociated with a reagent cartridge. The processor 1950 may control thetiming, duration, temperature, and other operations (including, e.g.,dispensing reagents) of the various modules of the system 1900 asspecified by one or more scripts. In addition to or as an alternative tothe one or more scripts, the processor may be programmed with standardprotocol parameters from which a user may select; alternatively, a usermay select one or all parameters manually. The script may specify, e.g.,the wavelength at which OD is read in the cell growth module, the targetOD to which the cells are grown, and the target time at which the cellswill reach the target OD. The processor may notify the user (e.g., viaan application to a smart phone or other device) that the cells havereached the target OD as well as update the user as to the progress ofthe cells in the cell growth module, electroporation device, filtrationmodule, recovery module, etc. in the automated multi-module cellprocessing system.

A second embodiment of an automated multi-module cell processing systemis shown in FIG. 20. As with the embodiment shown in FIG. 19, the cellprocessing system 2000 may include a housing 2060, a reservoir of cellsin, e.g., the reagent cartridge, where the cells are to be transformedor transfected 2002, and a growth module (a cell growth device) 2004.The cells to be transformed are transferred from, e.g., a reservoir inthe reagent cartridge to the growth module to be cultured until thecells hit a target OD. Once the cells hit the target OD, the growthmodule may cool or freeze the cells for later processing or transfer thecells to a filtration module 2030 where the cells are renderedelectrocompetent and concentrated to a volume optimal for celltransformation as described above in relation to FIG. 19. Onceconcentrated, the cells are then transferred to the FTEP device 2008 fortransformation.

In addition to the reservoir for storing the cells, the reagentcartridge may include a reservoir for storing editing oligonucleotides2016 and a reservoir for storing an expression vector backbone 2018.Both the editing oligonucleotides and the expression vector backbone aretransferred from, e.g., a reagent cartridge to a nucleic acid assemblymodule 2020 (such as the nucleic acid assembly module described above),where the editing oligonucleotides are inserted into the expressionvector backbone. The assembled nucleic acids may be transferred into anoptional purification module 2022 for desalting and/or otherpurification procedures needed to prepare the assembled nucleic acidsfor transformation. Once the processes carried out by the purificationmodule 2022 are complete, the assembled nucleic acids are alsotransferred to the FTEP device in reagent cartridge 2008, which alreadycontains the cell culture grown to a target OD, filtered and renderedelectrocompetent. In FTEP device 2008, the nucleic acids are introducedinto the cells. Following electroporation, the cells are transferredinto a combined recovery and editing module 2010. As described above, insome embodiments the automated multi-module cell processing system 2000is a system that performs gene editing such as an RNA-direct nucleaseediting system. For example, see U.S. Ser. No. 16/024,816, filed 30 Jun.2018; U.S. Ser. No. 16/024,831, filed 30 Jun. 2018; U.S. Ser. No.62/566,688, filed 2 Oct. 2017; and U.S. Ser. No. 62/567,698, filed 3Oct. 2017. In the recovery and editing module 2010, the cells areallowed to recover post-transformation, and the cells express theediting oligonucleotides that edit desired genes in the cells asdescribed below.

Following editing, the cells are transferred to a storage module 2012,where the cells can be stored at, e.g., 4° C. until the cells areretrieved for further study. The multi-module cell processing system iscontrolled by a processor 2050 configured to operate the instrumentbased on user input, as directed by one or more scripts, or as acombination of user input or a script. The processor 2050 may controlthe timing, duration, temperature, and operations of the various modulesof the system 2000 and the dispensing of reagents from, e.g., a reagentcartridge. The processor may be programmed with standard protocolparameters from which a user may select, a user may specify one or moreparameters manually or one or more scripts associated with the reagentcartridge may specify one or more operations and/or reaction parameters.In addition, the processor may notify the user (e.g., via an applicationto a smart phone or other device) that the cells have reached the targetOD as well as update the user as to the progress of the cells in thevarious modules in the multi-module system.

Certain embodiments of the multi-module processing system such as thesystem depicted in FIG. 20 include a nucleic acid assembly module (forexample, an assembly module that promotes gap repair in yeast and/or amodule that performs the Gibson Assembly™ reaction, polymerase chainreaction, ligation chain reaction, ligase detection reaction, ligation,circular polymerase extension cloning, or other assembly or cloningmethods) 2020. The nucleic acid assembly module 2020 is configured toassemble the nucleic acids necessary to facilitate genome editingevents. In a nuclease-directed genome editing system, a vector comprisesone or more regulatory elements operably linked to a polynucleotidesequence encoding a nucleic acid-guided nuclease. Thus, the nucleic acidassembly module 2020 in these embodiments is configured to assemble theexpression vector expressing a nucleic acid guided nuclease. The nucleicacid assembly module 2020 may be temperature controlled depending uponthe type of nucleic acid assembly used in the instrument. For example,when a nucleic acid assembly protocol is utilized, the module isconfigured to have the ability to reach and hold 50° C. If PCR isperformed as part of the automated multi-module cell processing system,the nucleic acid assembly module is configured to thermocycle betweentemperatures. The temperatures and duration for maintaining temperaturescan be controlled by a preprogrammed set of parameters (as dictated by ascript or programmed into the processor), or manually controlled by theuser using the processor.

As described above, in one embodiment the automated multi-module cellprocessing system 2000 is a nuclease-directed genome editing system.Multiple nuclease-based systems exist for providing edits into a cell,and each can be used in either single editing systems as could beperformed in the automated system 1900 of FIG. 19; sequential editingsystems as could be performed in the automated system 2100 of FIG. 21described below, e.g., using different nuclease-directed systemssequentially to provide two or more genome edits in a cell; and/orrecursive editing systems as could be performed in the automated system2100 of FIG. 21, e.g. utilizing a single nuclease-directed system tointroduce two or more genome edits in a cell simultaneously andsequentially. Automated nuclease-directed processing systems use thenucleases to cleave the cell's genome, to introduce one or more editsinto a target region of the cell's genome, or both. Nuclease-directedgenome editing mechanisms include zinc-finger editing mechanisms (seeUrnov et al., Nature Reviews Genetics, 11:636-64 (2010)), meganucleaseediting mechanisms (see Epinat et al., Nucleic Acids Research,31(11):2952-62 (2003); and Arnould et al., Journal of Molecular Biology,371(1):49-65 (2007)), and RNA-guided editing mechanisms (see Jinek etal., Science, 337:816-21 (2012); and Mali et al, Science, 339:823-26(2013)). In particular embodiments, the nuclease editing system is aninducible system that allows control of the timing of the editing (seeCampbell, Biochem J., 473(17): 2573-2589 (2016); and Dow et al., NatureBiotechnology, 33390-94 (2015)). That is, when the cell or population ofcells comprising a nucleic acid-guided nuclease encoding DNA is in thepresence of the inducer molecule, expression of the nuclease can occur.The ability to modulate nuclease activity can reduce off-target cleavageand facilitate precise genome engineering.

A third embodiment of a multi-module cell processing system is shown inFIG. 21. This embodiment depicts an exemplary system that performsrecursive gene editing on a cell population. As with the embodimentshown in FIGS. 19 and 20, the cell processing system 2100 may include ahousing 2160, a reservoir in, e.g., a reagent cartridge for storingcells to be transformed or transfected 2102, and a cell growth module (acell growth device) 2104. The cells to be transformed are transferredfrom a reservoir in the reagent cartridge to the cell growth module tobe cultured until the cells hit a target OD. Once the cells hit thetarget OD, the growth module may cool or freeze the cells for laterprocessing or the growth module may transfer the cells to a filtrationmodule 2120 where the cells are rendered electrocompetent, and thevolume of the cells may be reduced substantially. Once the cells havebeen concentrated to an appropriate volume, the cells are transferred toFTEP device 2108 in the reagent cartridge. In addition to the reservoirfor storing cells, the multi-module cell processing system includes areservoir for storing the vector comprising editing oligonucleotides2106 (that is, in this embodiment, the automated multi-module cellprocessing system does not comprise a nucleic acid assembly module;instead, the nucleic acids are provided pre-assembled). The assemblednucleic acids are transferred to the FTEP device 2108, which alreadycontains the cell culture grown to a target OD. In the FTEP device 2108,the nucleic acids are electroporated into the cells. Followingelectroporation, the cells are transferred into a recovery module 2124.In the recovery module 2124, the transformed cells are allowed torecover post-transformation.

The cells are transferred to a storage module 2112, where the cells canbe stored at, e.g., 4° C. until the cells are retrieved for furtherstudy, or the cells may be transferred to a second, optional, growthmodule 2126. Once the cells hit a target OD, the second growth modulemay cool or freeze the cells for later processing, or transfer the cellsto, e.g., an editing module 2128 where, e.g., one or both of aninducible nuclease and an inducible guide nucleic acid is activated inthe cells, e.g., by introduction of heat or the introduction of aninducer molecule for expression of the nuclease and/or guide nucleicacid. After editing, the cells are transferred to a separation andfiltration module 2130 where the cells are separated and/or concentratedfrom the editing solution in preparation for transfer to FTEP device2108.

In FTEP device 2108, the cells are transformed by a second set ofediting oligos (or other type of oligos) and the cycle is repeated untilthe cells have been transformed and edited by a desired number ofediting oligonucleotides. As discussed above in relation to FIGS. 19 and20, the multi-module cell processing system is controlled by a processor2150 configured to operate the instrument based on user input or iscontrolled by one or more scripts including at least one scriptassociated with the reagent cartridge. The processor 2150 may controlthe timing, duration, and temperature of various processes, thedispensing of reagents, and other operations of the various modules ofthe system 2100. For example, a script or the processor may control thedispensing of cells, reagents, vectors, and editing oligonucleotides;which editing oligonucleotides are used for cell editing and in whatorder; the time, temperature and other conditions used in the recoveryand expression module, the wavelength at which OD is read in the cellgrowth module, the target OD to which the cells are grown, and thetarget time at which the cells will reach the target OD. In addition,the processor may be programmed to notify a user (e.g., via anapplication) as to the progress of the cells in the automatedmulti-module cell processing system.

FIG. 22 is a simplified block diagram of an embodiment of an exemplaryautomated multi-module cell processing system comprising a singulationmodule for screening for edited cells. The cell processing system 2200may include a housing 2260, a reservoir of cells to be transformed ortransfected 2202, and a growth module (a cell growth device) 2204. Thecells to be transformed are transferred from a reservoir to the growthmodule to be cultured until the cells hit a target OD. Once the cellshit the target OD, the growth module may cool or freeze the cells forlater processing, or the cells may be transferred to an optionalfiltration module 2230 where the cells are rendered electrocompetent andconcentrated to a volume optimal for cell transformation. Onceconcentrated, the cells are then transferred to FTEP device 2208(transformation/transfection). Exemplary electroporation devices of usein the automated multi-module cell processing systems disclosed hereininclude those described in U.S. Ser. No. 62/566,374, filed 30 Sep. 2017;U.S. Ser. No. 62/556,375, filed 30 Sep. 2017; U.S. Ser. No. 62/657,651,filed 13 Apr. 2018; and U.S. Ser. No. 62/657,654, filed 13 Apr. 2018,all of which are herein incorporated by reference in their entirety.

In addition to the reservoir for storing the cells, the system 2200 mayinclude a reservoir for storing editing oligonucleotides 2216 and areservoir for storing an expression vector backbone 2218. Both theediting oligonucleotides and the expression vector backbone aretransferred from, e.g., reservoirs in a reagent cartridge to a nucleicacid assembly module 2220, where the editing oligonucleotides areinserted into the expression vector backbone. The assembled nucleicacids may be transferred into an optional purification module 2222 fordesalting and/or other purification procedures needed to prepare theassembled nucleic acids for transformation. Alternatively, pre-assemblednucleic acids, e.g., the editing vector, may be stored within reservoir2216 or 2218. Once the processes carried out by the purification module2222 are complete, the assembled nucleic acids are transferred to, e.g.,FTEP device 2208, which already contains the cell culture grown to atarget OD. In FTEP device 2208 the assembled nucleic acids areintroduced into the cells. Following electroporation, the cells aretransferred into a combined recovery, cell growth, and editing module2210. In some embodiments the automated multi-module cell processingsystem 2200 is a system that performs gene editing such as an RNA-directnuclease editing system. For example, see U.S. Ser. Nos. 16/024,816 and16/024,831, filed 30 Jun. 2018; U.S. Ser. No. 62/566,688, filed 2 Oct.2017; and U.S. Ser. No. 62/567,698, filed 3 Oct. 2017, all of which areherein incorporated by reference in their entirety. In the combinedrecovery, cell growth, and editing module 2210, the cells are allowed torecover post-transformation and editing commences.

Following editing, the cells are transferred to a singulation module2240, where the cells are arrayed such that there is an average of onecell per compartment. In some embodiments, a compartment may be a well,in some embodiments the compartment may be a droplet, and in someembodiments the compartment may be an area, e.g., cells isolated fromone another on an agar plate or arrayed on a functionalized substrate.Once singulated, the cells are allowed to grow and establish colonieswhich are grown to terminal size or saturation, limited by, e.g.,nutrients or physical confinement. Once colonies are established, thecolonies are pooled. Singulation overcomes growth bias from uneditedcells and growth bias resulting from fitness effects of different edits.

Once the cell colonies are pooled, the cells may be stored, e.g., in astorage module 2212, where the cells can be kept at, e.g., 4° C. untilthe cells are retrieved for further study. Alternatively, the cells maybe used in another round of editing. The multi-module cell processingsystem is controlled by a processor 2250 configured to operate theinstrument based on user input, as directed by one or more scripts, oras a combination of user input or a script. The processor 2250 maycontrol the timing, duration, temperature, and operations of the variousmodules of the system 2200 and the dispensing of reagents. For example,the processor 2250 may cool the cells post-transformation until editingis desired, upon which time the temperature may be raised to atemperature conducive of genome editing and cell growth. The processormay be programmed with standard protocol parameters from which a usermay select, a user may specify one or more parameters manually or one ormore scripts associated with the reagent cartridge may specify one ormore operations and/or reaction parameters. In addition, the processormay notify the user (e.g., via an application to a smart phone or otherdevice) that the cells have reached the target OD as well as update theuser as to the progress of the cells in the various modules in themulti-module system.

The automated multi-module cell processing system 2200 is anuclease-directed genome editing system and can be used in singleediting systems. The system of FIG. 23, described below, is configuredto perform sequential editing, e.g., using different nuclease-directedsystems sequentially to provide two or more genome edits in a cell;and/or recursive editing, e.g. utilizing a single nuclease-directedsystem to introduce two or more genome edits in a cell.

FIG. 23 illustrates another embodiment of a multi-module cell processingsystem. This embodiment depicts an exemplary system that 1) includesediting induction and cell selection in addition to screening, and 2)performs recursive gene editing on a cell population. As with theembodiment shown in FIG. 22, the cell processing system 2300 may includea housing 2360, a reservoir for storing cells to be transformed ortransfected 2302, and a cell growth module (a cell growth device) 2304.The cells to be transformed are transferred from a reservoir to the cellgrowth module to be cultured until the cells hit a target OD. Once thecells hit the target OD, the growth module may cool or freeze the cellsfor later processing or transfer the cells to an optional filtrationmodule 2330 where the cells are rendered electrocompetent, and thevolume of the cells may be reduced substantially. Once the cells havebeen concentrated to an appropriate volume, the cells are transferred toFTEP device 2308. In addition to the reservoir for storing cells, themulti-module cell processing system includes a reservoir for storing thevector comprising editing oligonucleotides 2352. The assembled nucleicacids are transferred to FTEP device 2308, which already contains thecell culture grown to a target OD. In the FTEP device 2308, the nucleicacids are electroporated into the cells. Following electroporation, thecells are transferred into an optional recovery module 2342, where thecells are allowed to recover briefly post-transformation.

After recovery, the cells may be transferred to a storage module 2312,where the cells can be stored at, e.g., 4° C. until the cells areretrieved for further study, or the cells may be transferred to asingulation and growth module 2344. In the singulation module 2344, thecells are arrayed such that there is an average of one cell percompartment. In some embodiments, a compartment may be a well; adroplet; or an area, e.g., cells isolated from one another on an agarplate or arrayed on, e.g., a functionalized substrate. Once singulated,the cells are allowed to grow through several to many doublings andestablish colonies. Once colonies are established, the substrate withthe cell colonies is transferred to an induction module 2346, whereconditions exist (temperature, addition of an inducing or repressingchemical) to induce editing. Once editing is initiated and allowed toproceed, the substrate is transferred to a selection module 2348, whichmay include, e.g., a colony measuring and picking device that selectssmall colonies of cells; a spectrophotometer configured to measure OD inwells or droplets and collect colonies of edited cells based on cellgrowth; or a spectrophotometer configured to measure other cellularcharacteristics in wells or droplets and collect colonies of editedcells based on cell characteristics that correlate with cell growth.Note that the singulation module and selection module may be linked.Once the putatively-edited cells are selected, they may be subjected toanother round of editing, beginning with transformation by yet anotherdonor nucleic acid in another editing cassette via the FTEP module 2308.

In FTEP device 2308, the cells selected from the first round of editingare transformed by a second set of editing oligos (or other type ofoligos) and the cycle is repeated until the cells have been transformedand edited by a desired number of, e.g., donor nucleic acids. Themulti-module cell processing system exemplified in FIG. 23 is controlledby a processor 2350 configured to operate the instrument based on userinput, or is controlled by one or more scripts including at least onescript associated with the reagent cartridge. The processor 2350 maycontrol the timing, duration, and temperature of various processes, thedispensing of reagents, and other operations of the various modules ofthe system 2300. For example, a script or the processor may control thedispensing of cells, reagents, vectors, and editing oligonucleotides;which editing oligonucleotides are used for cell editing and in whatorder; the time, temperature and other conditions used in the recoveryand expression module, the wavelength at which OD is read in the cellgrowth module, the target OD to which the cells are grown, and thetarget time at which the cells will reach the target OD. In addition,the processor may be programmed to notify a user (e.g., via anapplication) as to the progress of the cells in the automatedmulti-module cell processing system.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention, nor are theyintended to represent or imply that the experiments below are all of orthe only experiments performed. It will be appreciated by personsskilled in the art that numerous variations and/or modifications may bemade to the invention as shown in the specific aspects without departingfrom the spirit or scope of the invention as broadly described. Thepresent aspects are, therefore, to be considered in all respects asillustrative and not restrictive.

Example 1: Production and Transformation of Electrocompetent E. coli

For testing transformation of the FTEP device, electrocompetent E. colicells were created. To create a starter culture, 6 ml volumes of LBchlor-25 (LB with 25 μg/ml chloramphenicol) were transferred to 14 mlculture tubes. A 25 μl aliquot of E. coli was used to inoculate the LBchlor-25 tubes. Following inoculation, the tubes were placed at a 45°angle in the shaking incubator set to 250 RPM and 30° C. for overnightgrowth, between 12-16 hrs. The OD600 value should be between 2.0 and4.0. A 1:100 inoculum volume of the 250 ml LB chlor-25 tubes weretransferred to four sterile 500 ml baffled shake flasks, i.e., 2.5 mlper 250 ml volume shake flask. The flasks were placed in a shakingincubator set to 250 RPM and 30° C. The growth was monitored bymeasuring OD600 every 1 to 2 hr. When the OD600 of the culture wasbetween 0.5-0.6 (approx. 3-4 hrs), the flasks were removed from theincubator. The cells were centrifuged at 4300 RPM, 10 min, 4° C. Thesupernatant was removed, and 100 ml of ice-cold 10% glycerol wastransferred to each sample. The cells were gently resuspended, and thewash procedure performed three times, each time with the cellsresuspended in 10% glycerol. After the fourth centrifugation, the cellresuspension was transferred to a 50 ml conical Falcon tube andadditional ice-cold 10% glycerol added to bring the volume up to 30 ml.The cells were again centrifuged at 4300 RPM, 10 min, 4° C., thesupernatant removed, and the cell pellet resuspended in 10 ml ice-coldglycerol. The cells are aliquoted in 1:100 dilutions of cell suspensionand ice-cold glycerol.

The comparative electroporation experiment was performed to determinethe efficiency of transformation of the electrocompetent E. coli usingthe embodiment of the FTEP device shown at (ii), (iii), and (vi) ofFIGS. 15B and 15C and (ii) and (vi) of FIG. 15D. (See FIG. 24 for ascanning electromicrograph demonstrating laminar flow of cells andexogenous material through the narrowed portion of the flow channel ofan FTEP device). The flow rate was controlled with a pressure controlsystem. The suspension of cells with DNA was loaded into the FTEP inletreservoir. The transformed cells flowed directly from the inlet andinlet channel, through the flow channel, through the outlet channel, andinto the outlet containing recovery medium. The cells were transferredinto a tube containing additional recovery medium, placed in anincubator shaker at 30° C. shaking at 250 rpm for 3 hours. The cellswere plated to determine the colony forming units (CFUs) that survivedelectroporation and failed to take up a plasmid and the CFUs thatsurvived electroporation and took up a plasmid. Plates were incubated at30° C.; E. coli colonies were counted after 24 hrs.

The flow-through electroporation experiments were benchmarked against 2mm electroporation cuvettes (Bull dog Bio) using an in vitro highvoltage electroporator (NEPAGENE™ ELEPO21). Stock tubes of cellsuspensions with DNA were prepared and used for side-to-side experimentswith the NEPAGENE™ and the flow-through electroporation. The results areshown in FIG. 25A. In FIG. 25A, the left-most bars hatched /// denotecell input, the bars to the left bars hatched \\\ denote the number ofcells that survived transformation, and the right bars hatched ///denote the number of cells that were actually transformed. The FTEPdevice showed equivalent transformation of electrocompetent E. colicells at various voltages as compared to the NEPAGENE™ electroporator.As can be seen, the transformation survival rate is at least 90% and insome embodiments is at least 95%, 96%, 97%, 98%, or 99%. The recoveryratio (the fraction of introduced cells which are successfullytransformed and recovered) is in certain embodiments at least 0.001 andpreferably between 0.00001 and 0.01. In FIG. 25A the recovery ratio isapproximately 0.0001.

Additionally, a comparison of the NEPAGENE™ ELEPO21 and the FTEP devicewas made for efficiencies of transformation (uptake), cutting, andediting. In FIG. 25B, triplicate experiments were performed where thebars hatched /// denote the number of cells input for transformation,and the bars hatched \\\ denote the number of cells that weretransformed (uptake), the number of cells where the genome of the cellswas cut by a nuclease transcribed and translated from a vectortransformed into the cells (cutting), and the number of cells whereediting was effected (cutting and repair using a nuclease transcribedand translated from a vector transformed into the cells, and using aguide RNA and a donor DNA sequence both of which were transcribed from avector transformed into the cells). Again, it can be seen that the FTEPshowed equivalent transformation, cutting, and editing efficiencies asthe NEPAGENE™ electroporator. The recovery rate in FIG. 25B for the FTEPis treater than 0.001.

Example 2: Production and Transformation of Electrocompetent S.Cerevisiae

For further testing transformation of the FTEP device, S. Cerevisiaecells were created using the methods as generally set forth inBergkessel and Guthrie, Methods Enzymol., 529:311-20 (2013). Briefly,YFAP media was inoculated for overnight growth, with 3 ml inoculate toproduce 100 ml of cells. Every 100 ml of culture processed resulted inapproximately 1 ml of competent cells. Cells were incubated at 30° C. ina shaking incubator until they reached an OD600 of 1.5+/−0.1.

A conditioning buffer was prepared using 100 mM lithium acetate, 10 mMdithiothreitol, and 50 mL of buffer for every 100 mL of cells grown andkept at room temperature. Cells were harvested in 250 ml bottles at 4300rpm for 3 minutes, and the supernatant removed. The cell pellets weresuspended in 100 ml of cold 1 M sorbitol, spun at 4300 rpm for 3 minutesand the supernatant once again removed. The cells were suspended inconditioning buffer, then the suspension transferred into an appropriateflask and shaken at 200 RPM and 30° C. for 30 minutes. The suspensionswere transferred to 50 ml conical vials and spun at 4300 rpm for 3minutes. The supernatant was removed and the pellet resuspended in cold1 M sorbitol. These steps were repeated three times for a total of threewash-spin-decant steps. The pellet was suspended in sorbitol to a finalOD of 150+/−20.

A comparative electroporation experiment was performed to determine theefficiency of transformation of the electrocompetent S. Cerevisiae usingthe FTEP device. The flow rate was controlled with a syringe pump(Harvard apparatus PHD ULTRA™ 4400). The suspension of cells with DNAwas loaded into a 1 mL glass syringe (Hamilton 81320 Syringe, PTFE LuerLock) before mounting on the pump. The output from the functiongenerator was turned on immediately after starting the flow. Theprocessed cells flowed directly into a tube with 1M sorbitol withcarbenicillin. Cells were collected until the same volume electroporatedin the NEPAGENE™ had been processed, at which point the flow and theoutput from the function generator were stopped. After a 3-hour recoveryin an incubator shaker at 30° C. and 250 rpm, cells were plated todetermine the colony forming units (CFUs) that survived electroporationand failed to take up a plasmid and the CFUs that survivedelectroporation and took up a plasmid. Plates were incubated at 30° C.Yeast colonies are counted after 48-76 hrs.

The flow-through electroporation experiments were benchmarked against 2mm electroporation cuvettes (Bull dog Bio) using an in vitro highvoltage electroporator (NEPAGENE™ ELEPO21). Stock tubes of cellsuspensions with DNA were prepared and used for side-to-side experimentswith the NEPAGENE™ and the flow-through electroporation. The results areshown in FIG. 26. The device showed better transformation and survivalof electrocompetent S. Cerevisiae at 2.5 kV voltages as compared to theNEPAGENE™ method. Input is total number of cells that were processed.

Example 3: FTEP Pressure Sensing and Flow Rates

An inline flow sensor measurement was used to indicate when, after theliquid containing the cells and DNA flowed through the FTEP chip, wherethe inlet reservoir was emptied. Approximately 65 μL of liquid wasloaded into the input reservoir and the automated FTEP module waspowered on. Looking at the graph at the top of FIG. 27, it can be seenthat after a few short startup transients, the flow rate shows about ˜3standard cubic centimeters per minute (SCCM) of flow for almost 8seconds (8000 ms) until it jumps to 24 SCCM. This transition occurs atan end of run trigger, which is an indicator that the liquid containingthe cells and DNA has been processed through the FTEP device and thatair is not flowing through the FTEP device. That trigger may constitutedetection of an increase flow rate or a sudden fluctuation (increase ordecrease) in the pressure of the air (such as at a conduit leading froma syringe pump). In one preferred embodiment, the flow sensor in FIG. 27detects an increase in air flow indicative of the fluid being completelydrained from the input reservoir. At this point, pressure may bereversed to allow a multi-pass electroporation procedure; that is, cellsto electroporated may be “pulled” from the inlet toward the outlet forone pass of electroporation, and once the inlet reservoir is emptied,the sensor may reverse the pressure where the liquid and cells/DNA is“pushed” from the outlet end of the flow-through FTEP device toward theinlet end to pass between the electrodes again for another pass ofelectroporation. This process may be repeated one to many times.Alternatively, the pressure may be stopped entirely and the transformedcells in the outlet retrieved.

The multi-cycle approach may be particularly advantageous in that itlimits the dwell time of the cells and exogenous materials in theelectric filed which may in turn prevent cell damage and increasesurvival rates. The back-and-forth process may be repeated 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 times. FIG. 27 at bottom shows a simple depictionof the pressure system and FTEP. The pressure manifold is mated to theupwardly-extending reservoirs via one or more complementary seals orgaskets disposed on the manifold or the reservoirs. The manifolds maytake the form of the “lids” 1608 shown in FIG. 16B (v).

While this invention is satisfied by embodiments in many differentforms, as described in detail in connection with preferred embodimentsof the invention, it is understood that the present disclosure is to beconsidered as exemplary of the principles of the invention and is notintended to limit the invention to the specific embodiments illustratedand described herein. Numerous variations may be made by persons skilledin the art without departure from the spirit of the invention. The scopeof the invention will be measured by the appended claims and theirequivalents. The abstract and the title are not to be construed aslimiting the scope of the present invention, as their purpose is toenable the appropriate authorities, as well as the general public, toquickly determine the general nature of the invention. In the claimsthat follow, unless the term “means” is used, none of the features orelements recited therein should be construed as means-plus-functionlimitations pursuant to 35 U.S.C. § 112, ¶6.

1. A flow-through electroporation (FTEP) device for introducing anexogenous material into cells in a fluid, the FTEP device comprising: a.at least a first inlet and at least a first inlet channel for receivinga fluid comprising cells and/or exogenous material into the FTEP device;b. an outlet and an outlet channel for removing a fluid comprisingtransformed cells and exogenous material from the FTEP device; c. a flowchannel intersecting and positioned between the at least first inletchannel and the outlet channel, wherein the flow channel decreases inwidth to a dimension no smaller than at least 2× diameter of the cellsbeing electroporated between the first inlet channel and a center of theflow channel and the outlet channel and the center of the flow channel;and d. two electrodes positioned in electrode channels between theintersection of the flow channel with the first inlet channel and theintersection of the flow channel with the outlet channel and on eitherend of where the flow channel decreases in width; wherein the electrodesare in fluid and electrical communication with fluid in the flow channelbut not directly in the path of the cells traveling through the flowchannel; and wherein the electrodes apply one or more electric pulses tothe cells in the fluid as they pass through the flow channel, therebyintroducing exogenous material into the cells in the fluid.
 2. The FTEPdevice of claim 1, further comprising a reservoir connected to the inletfor introducing the cells in fluid into the FTEP device and a reservoirconnected to the outlet for removing transformed cells from the FTEPdevice.
 3. The FTEP device of claim 2, wherein the FTEP device comprisestwo inlets and two inlet channels and further comprising a reservoirconnected to a second inlet for introducing exogenous material into theFTEP device.
 4. The FTEP device of claim 3, wherein the second inlet andsecond inlet channel are located between the first inlet and first inletchannel and the electrodes.
 5. The FTEP device of claim 3, wherein thesecond inlet and second inlet channel are located between the electrodesand the outlet channel.
 6. The FTEP device of claim 1, wherein device isconfigured for use with bacterial, yeast and mammalian cells.
 7. TheFTEP device of claim 1, wherein the two electrodes are located from 1 mmto 10 mm from one another.
 8. The FTEP device of claim 1, wherein thenarrowest part of the channel width is from 10 μM to 5 mm.
 9. The FTEPdevice of claim 1, wherein the device further comprises one or morefilters between the one or more inlet channels and the outlet channel.10. An automated multi-module cell processing system, comprising theFTEP device of claim
 1. 11. A flow-through electroporation (FTEP) devicefor introducing an exogenous material into cells in a fluid, the devicecomprising: at least one inlet and at least one inlet channel forintroducing a fluid comprising cells and exogenous material to the FTEPdevice; an outlet and an outlet channel for removing transformed cellsand exogenous material from the FTEP device; a flow channel positionedbetween a first inlet channel and the outlet channel, wherein the flowchannel intersects with the first inlet channel and the outlet channeland wherein a portion of the flow channel narrows between the inletchannel intersection and the outlet channel intersection to a dimensionno smaller than at least 2× diameter of the cells being electroporated;and an electrode positioned on either side of the flow channel and indirect contact with the fluid in the flow channel, the electrodesdefining the narrowed portion of the flow channel; and wherein theelectrodes apply one or more electric pulses to the cells in the fluidas they pass through the flow channel, thereby introducing the exogenousmaterial into the cells in the fluid.
 12. The FTEP device of claim 11,further comprising a reservoir connected to the inlet for introducingthe cells in fluid into the FTEP device and a reservoir connected to theoutlet for removing transformed cells from the FTEP device.
 13. The FTEPdevice of claim 12, wherein the FTEP device comprises two inlets and twoinlet channels and further comprising a reservoir connected to a secondinlet for introducing the exogenous material into the FTEP device. 14.The FTEP device of claim 13, wherein the second inlet and second inletchannel are located between the first inlet and first inlet channel andthe electrodes.
 15. The FTEP device of claim 13, wherein the secondinlet and second inlet channel are located between the electrodes andthe outlet channel and outlet.
 16. The FTEP device of claim 11, whereindevice is configured for use with bacterial, yeast and mammalian cells.17. The FTEP device of claim 11, wherein the two electrodes are from 10μm to 1 mm from one another.
 18. The FTEP device of claim 11, whereinthe narrowest part of the channel width is from 10 μM to 1 mm.
 19. TheFTEP device of claim 11, wherein the device further comprises one ormore filters between the at least one inlet channel and the outletchannel.
 20. An automated multi-module cell processing system comprisingthe FTEP device of claim 11.